Nutritive Fragments, Proteins and Methods

ABSTRACT

Nutritive proteins are provided. In some embodiments the nutritive proteins comprise a first polypeptide sequence comprising a fragment of a naturally-occurring nutritive protein. In some embodiments the fragment comprises at least one of a) an enhanced ratio of branch chain amino acid residues to total amino acid residues present in the nutritive protein; b) an enhanced ratio of leucine residues to total amino acid residues present in the nutritive protein; and c) an enhanced ratio of essential amino acid residues to total amino acid residues present in the nutritive protein. In some embodiments, the fragment comprises at least one of a) a ratio of branch chain amino acid residues to total amino acid residues present in the nutritive protein; b) a ratio of leucine residues to total amino acid residues present in the nutritive protein; and c) a ratio of essential amino acid residues to total amino acid residues present in the nutritive protein, that is equal to or greater than the corresponding ratio present in a benchmark protein such as whey, egg or soy protein. Also provided are nucleic acids encoding the proteins, recombinant microorganisms that make the proteins, methods of making the proteins using recombinant microorganisms, compositions that comprise the proteins, and methods of using the proteins, among other things.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/374,127, filed on Jul. 23, 2014, which is a 371 application thatclaims priority to PCT/US2013/032218, filed Mar. 15, 2013, which claimspriority to U.S. Provisional Patent Application No. 61/615,819, filedMar. 26, 2012, each of which is hereby incorporated herein by referencein its entirety, for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 9, 2017, isnamed 36280_US_Sequence_Listings.txt and is 3,234,691 bytes in size.

INTRODUCTION

Dietary protein is an essential nutrient for human health and growth.The World Health Organization recommends that dietary protein shouldcontribute approximately 10 to 15% of energy intake when in energybalance and weight stable. Average daily protein intakes in variouscountries indicate that these recommendations are consistent with theamount of protein being consumed worldwide. Meals with an average of 20to 30% of energy from protein are representative of high-protein dietswhen consumed in energy balance.

The body cannot synthesize certain amino acids that are necessary forhealth and growth, and instead must obtain them from food. These aminoacids, called “essential amino acids”, are Histidine (H), Isoleucine(I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F),Threonine (T), Tryptophan (W), and Valine (V). Dietary proteins thatprovide all the essential amino acids are referred to as “high quality”proteins. Animal foods such as meat, fish, poultry, eggs, and dairyproducts are generally regarded as high quality protein sources thatprovide a good balance of essential amino acids. Casein (a proteincommonly found in mammalian milk, making up 80% of the proteins in cowmilk) and whey (the protein in the liquid that remains after milk hasbeen curdled and strained) are major sources of high quality dietaryprotein. Foods that do not provide a good balance of essential aminoacids are referred to as “low quality” proteins. Most fruits andvegetables are poor sources of protein. Some plants foods includingbeans, peas, lentils, nuts and grains (such as wheat) are better sourcesof protein. Soy, a vegetable protein manufactured from soybeans, isconsidered by some to be a high quality protein.

Studies of the acute effects of consuming high amounts of protein inhumans have shown that inclusion and in some cases increasing proteincontent in the diet can have beneficial effects. For example, studieshave shown that ingestion of protein can induce postprandial satiety(including by suppressing hunger), induce thermogenesis and reduceglycemic response in human subjects.

Studies of high protein diets for weight loss have shown that proteinpositively affects energy expenditure and lean body mass. Furtherstudies have shown that overeating produces significantly less weightgain in diets containing at least 5% of energy from protein, and that ahigh-protein diet decreases energy intake.

Clinical studies provide evidence that protein prevents muscle loss dueto aging or bed rest. In particular, studies have shown that proteinsupplementation increases muscle fractional synthetic rate (FSR) duringprolonged bed rest, maintains leg mass and strength during prolonged bedrest increases lean body mass, improves functional measures of gait andbalance, and may serve as a viable intervention for individuals at riskof sarcopenia due to immobility or prolonged bed rest.

Studies on increasing muscle protein anabolism in athletes have shownthat protein provided following exercise promotes muscle hypertrophy toa greater extent than that achieved by exercise alone. It has also beenshown that protein provided following exercise supports proteinsynthesis without any increase in protein breakdown, resulting in a netpositive protein balance and muscle mass accretion. While muscle proteinsynthesis appears to respond in a dose-response fashion to essentialamino acid supplementation, not all proteins are equal in buildingmuscle. For example, milk proteins appear to be superior to soy insupporting muscle mass accretion with resistance training, while bothare superior to carbohydrate alone. The amino acid leucine is animportant factor in stimulating muscle protein synthesis.

Whole proteins commonly found in foods do not necessarily provide anamino acid composition that meets the amino acid requirements of amammal, such as a human, in an efficient manner. The result is that, inorder to attain the minimal requirements of each essential amino acid, alarger amount of total protein must be consumed in the diet than wouldbe required if the quality of the dietary protein were higher. Byincreasing the quality of the protein in the diet it is possible toreduce the total amount of protein that must be consumed compared todiets that include lower quality proteins.

In general, proteins that have higher protein quality are consideredmore beneficial in a mammalian diet than other proteins that do not.Such proteins are useful, for example, as components of a mammaliandiet. Under certain circumstances such proteins promote maintenance ofmuscle mass, a healthy body mass index, and glycemic balance, amongother things. Accordingly, there is a need for sources of proteins thathave high protein quality.

Traditionally, desirable mixtures of amino acids, such as mixturescomprising essential amino acids, have been provided by hydrolyzing aprotein with relatively high levels of essential amino acids, such aswhey protein, and/or by combining free amino acids in a mixture thatoptionally also includes a hydrolyzed protein such as whey. Mixtures ofthis type may have a bitter taste and may be deemed unsuitable orundesirable for certain uses. As a result, such mixtures sometimesinclude flavoring agents to mask the taste of the free amino acidsand/or hydrolyzed protein. In some cases compositions in which aproportion of the amino acid content is provided by polypeptides orproteins are found to have a better taste than compositions with a highproportion of total amino acids provided as free amino acids and/orcertain hydrolyzed proteins. The availability of such compositions hasbeen limited, however, because nutritional formulations havetraditionally been made from protein isolated from natural foodproducts, such as whey isolated from milk, or soy protein isolated fromsoy. The amino acid profiles of those proteins do not necessarily meetthe amino acid requirements for a mammal. In addition, commodityproteins typically consist of mixtures of proteins and/or proteinhydrolysates which can vary in their protein composition, thus leadingto unpredictability regarding their nutritional value. Moreover, thelimited number of sources of such high quality proteins has meant thatonly certain combinations of amino acids are available on a large scalefor ingestion in protein form.

The agricultural methods required to supply high quality animal proteinsources such as casein and whey, eggs, and meat, as well as plantproteins such as soy, also require significant energy inputs and havepotentially deleterious environmental impacts. Accordingly, it would beuseful in certain situations to have alternative sources and methods ofsupplying proteins for mammalian consumption.

In theory, synthetic polypeptide sequences comprising a desired mixtureof amino acids could be designed and produced in a laboratory setting.This approach may raise various concerns, however, and is therefore notalways applicable. First, skilled artisans are aware that obtaining highlevels of production of such synthetic sequences may be verychallenging. Second, even if such a synthetic protein were synthesized,its suitability for use in a nutritive product would be uncertain. Forexample, such a non-naturally occurring polypeptide could be an allergenor a toxin. Accordingly, in some embodiments this disclosure providesnatural protein or polypeptide sequences, or variants thereof.

In this disclosure the inventors provide nutritive proteins comprising afirst polypeptide sequence that is homologous to a fragment of anaturally-occurring protein. The nutritive proteins and the fragments ofwhich they are composed are made up of useful combinations of aminoacids. The proteins can be produced by methods that do not rely solelyon traditional agriculture for production. For example, the inventorshave discovered and this disclosure provides nutritive proteinscomprising a fragment of a naturally-occurring protein and composed ofcombinations of amino acids that contain a useful level of at least oneof a ratio of branch chain amino acids to total amino acids, a ratio ofthe amino acid leucine to total amino acids, and a ratio essential aminoacids to total amino acids. In some embodiments the nutritive proteinscomprise at least one of a level of a) a ratio of branch chain aminoacid residues to total amino acid residues present in the nutritiveprotein equal to or greater than the ratio of branch chain amino acidresidues to total amino acid residues present in whey protein; b) aratio of leucine residues to total amino acid residues present in thenutritive protein equal to or greater than the ratio of leucine residuesto total amino acid residues present in whey protein; and c) a ratio ofessential amino acid residues to total amino acid residues present inthe nutritive protein equal to or greater than the ratio of essentialamino acid residues to total amino acid residues present in wheyprotein.

This disclosure also provides nucleic acids encoding the proteins,recombinant microorganisms that make the proteins, methods of making theproteins using recombinant microorganisms, compositions that comprisethe proteins, and methods of using the proteins, among other things.

SUMMARY

In a first aspect this disclosure provides isolated nutritive proteinscomprising a first polypeptide sequence that is homologous to a fragmentof a naturally-occurring protein, wherein the first polypeptide sequencecomprises at least one of: a. a ratio of branch chain amino acidresidues to total amino acid residues of at least 24%; b. a ratio of Leuresidues to total amino acid residues of at least 11%; and c. a ratio ofessential amino acid residues to total amino acid residues of at least49%. In some embodiments the first polypeptide sequence furthercomprises at least one of each essential amino acid. In some embodimentsthe first polypeptide sequence comprises at least 70% homology to thefragment of a naturally-occurring protein. In some embodiments the firstpolypeptide sequence comprises at least 95% homology to the fragment ofa naturally-occurring protein. In some embodiments the fragment of anaturally-occurring protein comprises at least 25 amino acid residues.In some embodiments the fragment of a naturally-occurring proteincomprises at least 50 amino acid residues. In some embodiments the firstpolypeptide sequence comprises: a. a ratio of branch chain amino acidresidues to total amino acid residues of at least 24%; b. a ratio of Leuresidues to total amino acid residues of at least 11%; and c. a ratio ofessential amino acid residues to total amino acid residues of at least49%. In some embodiments the first polypeptide sequence comprises afragment of a naturally-occurring nutritive protein. In some embodimentsthe first polypeptide sequence consists of a fragment of anaturally-occurring nutritive protein.

In some embodiments the first polypeptide sequence is not an allergen.In some embodiments the first polypeptide sequence has less than 50%global homology to a known allergen.

In some embodiments the first polypeptide sequence is not a toxin. Insome embodiments the first polypeptide sequence has less than 50% globalhomology to a known toxin.

In some embodiments the first polypeptide sequence has a simulatedgastric digestion half-life of less than 60 minutes. In some embodimentsthe first polypeptide sequence has a simulated gastric digestionhalf-life of less than 30 minutes. In some embodiments the firstpolypeptide sequence has a simulated gastric digestion half-life of lessthan 10 minutes. In some embodiments the first polypeptide sequence iscompletely digested in simulated gastric fluid. In some embodiments thefirst polypeptide sequence comprises at least one protease recognitionsite selected from a pepsin recognition site, a trypsin recognitionsite, and a chymotrypsin recognition site. In some embodiments the firstpolypeptide sequence comprises no cysteine residues. In some embodimentsthe first polypeptide sequence comprises no disulfide bonds. In someembodiments the first polypeptide sequence does not comprise N-linkedglycosylation. In some embodiments the first polypeptide sequence doesnot comprise O-linked glycosylation.

In some embodiments the first polypeptide sequence is resistant toaggregation. In some embodiments the first polypeptide sequence isanionic at pH 7. In some embodiments the first polypeptide sequence hasan aqueous solubility at pH 7 of at least 12.5 g/L. In some embodimentsthe first polypeptide sequence has a calculated solvation score of −20or less. In some embodiments the first polypeptide sequence has acalculated aggregation score of 0.75 or less. In some embodiments thefirst polypeptide sequence has a calculated aggregation score of 0.5 orless.

In some embodiments the first polypeptide sequence comprises an aminoacid sequence selected from: i. an amino acid sequence selected from SEQID NO: 1 to SEQ ID NO: 2609; ii. a modified derivative of an amino acidsequence selected from SEQ ID NO: 1 to SEQ ID NO: 2609; and iii. amutein of an amino acid sequence selected from SEQ ID NO: 1 to SEQ IDNO: 2609. In some embodiments the first polypeptide sequence consists ofan amino acid sequence selected from: i. an amino acid sequence selectedfrom SEQ ID NO: 1 to SEQ ID NO: 2609; ii. a modified derivative of anamino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 2609; andiii. a mutein of an amino acid sequence selected from v. In someembodiments the first polypeptide sequence is at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to at least oneamino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 2609.

In some embodiments the first polypeptide sequence is a fragment of atleast 25 amino acids of the naturally-occurring nutritive protein. Insome embodiments the first polypeptide sequence is a fragment of atleast 50 amino acids of the naturally-occurring nutritive protein. Insome embodiments the isolated nutritive protein consists of the firstpolypeptide sequence. In some embodiments the isolated nutritive proteinfurther comprises a polypeptide tag for affinity purification. In someembodiments the tag for affinity purification is a polyhistidine-tag.

In another aspect this disclosure provides isolated nutritive proteinscomprising a first polypeptide sequence that is homologous to a fragmentof a naturally-occurring protein, wherein the isolated nutritive proteincomprises at least one of: a. a ratio of branch chain amino acidresidues to total amino acid residues of at least 24%; b. a ratio of Leuresidues to total amino acid residues of at least 11%; and c. a ratio ofessential amino acid residues to total amino acid residues of at least49%. In some embodiments the isolated nutritive protein furthercomprises at least one of each essential amino acid. In some embodimentsthe isolated nutritive protein comprises at least 70% homology to thefragment of a naturally-occurring protein. In some embodiments theisolated nutritive protein comprises at least 95% homology to thefragment of a naturally-occurring protein. In some embodiments thefragment of a naturally-occurring protein comprises at least 25 aminoacid residues. In some embodiments the fragment of a naturally-occurringprotein comprises at least 50 amino acid residues. In some embodimentsthe isolated nutritive protein comprises: a. a ratio of branch chainamino acid residues to total amino acid residues of at least 24%; b. aratio of Leu residues to total amino acid residues of at least 11%; andc. a ratio of essential amino acid residues to total amino acid residuesof at least 49%. In some embodiments the isolated nutritive proteincomprises a fragment of a naturally occurring nutritive protein. In someembodiments the isolated nutritive protein consists of a fragment of anaturally occurring nutritive protein.

In some embodiments the isolated nutritive protein is not an allergen.In some embodiments the isolated nutritive protein has less than 50%global homology to a known allergen.

In some embodiments the isolated nutritive protein is not a toxin. Insome embodiments the isolated nutritive protein has less than 50% globalhomology to a known toxin.

In some embodiments the isolated nutritive protein has a simulatedgastric digestion half-life of less than 60 minutes. In some embodimentsthe isolated nutritive protein has a simulated gastric digestionhalf-life of less than 30 minutes. In some embodiments the isolatednutritive protein has a simulated gastric digestion half-life of lessthan 10 minutes. In some embodiments the isolated nutritive protein iscompletely digested in simulated gastric fluid. In some embodiments theisolated nutritive protein comprises at least one protease recognitionsite selected from a pepsin recognition site, a trypsin recognitionsite, and a chymotrypsin recognition site. In some embodiments theisolated nutritive protein comprises no cysteine residues. In someembodiments the isolated nutritive protein comprises no disulfide bonds.In some embodiments the isolated nutritive protein does not compriseN-linked glycosylation. In some embodiments the isolated nutritiveprotein does not comprise O-linked glycosylation.

In some embodiments the isolated nutritive protein is resistant toaggregation. In some embodiments the isolated nutritive protein isanionic at pH 7. In some embodiments the isolated nutritive protein hasan aqueous solubility at pH 7 of at least 12.5 g/L. In some embodimentsthe isolated nutritive protein has a calculated solvation score of −20or less. In some embodiments the isolated nutritive protein has acalculated aggregation score of 0.75 or less. In some embodiments theisolated nutritive protein has a calculated aggregation score of 0.5 orless.

In some embodiments the isolated nutritive protein comprises an aminoacid sequence selected from: i. an amino acid sequence selected from SEQID NO: 1 to SEQ ID NO: 2609; ii. a modified derivative of an amino acidsequence selected from SEQ ID NO: 1 to SEQ ID NO: 2609; and iii. amutein of an amino acid sequence selected from SEQ ID NO: 1 to SEQ IDNO: 2609. In some embodiments the isolated nutritive protein consists ofan amino acid sequence selected from: i. an amino acid sequence selectedfrom SEQ ID NO: 1 to SEQ ID NO: 2609; ii. a modified derivative of anamino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 2609; andiii. a mutein of an amino acid sequence selected from SEQ ID NO: 1 toSEQ ID NO: 2609. In some embodiments the isolated nutritive protein isat least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%homologous to at least one amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO: 2609. In some embodiments the isolated nutritive proteinfurther comprises a polypeptide tag for affinity purification. In someembodiments the tag for affinity purification is a polyhistidine-tag.

In another aspect this disclosure provides isolated nucleic acidscomprising a nucleic acid sequence that encodes a nutritive proteinaccording to this disclosure. In some embodiments the isolated nucleicacid is selected from genomic DNA, cDNA, sense RNA and antisense RNA. Insome embodiments the isolated nucleic acid is genomic DNA. In someembodiments the isolated nucleic acid is cDNA. In some embodiments theisolated nucleic acid further comprises an expression control sequenceoperatively linked to the nucleic acid sequence that encodes thenutritive protein. In some embodiments the nucleic acid sequence thatencodes a nutritive protein according to this disclosure is present in avector. In some embodiments the vector further comprises an expressioncontrol sequence operatively linked to the nucleic acid sequence thatencodes the nutritive protein.

In another aspect this disclosure provides recombinant microorganismscomprising at least one of a nucleic acid and a vector according to thisdisclosure. In some embodiments the recombinant microorganism is aprokaryote. In some embodiments the prokaryote is heterotrophic. In someembodiments the prokaryote is autotrophic. In some embodiments theprokaryote is a bacteria.

In another aspect this disclosure provides methods of making a nutritiveprotein according to this disclosure, the method comprising culturing arecombinant microorganism according to this disclosure under conditionssufficient for production of the nutritive protein by the recombinantmicroorganism. In some embodiments the method further comprisesisolating the nutritive protein from the culture.

In another aspect this disclosure nutritive compositions comprising anutritive protein of this disclosure and at least one second component.In some embodiments the at least one second component is selected from aprotein, a polypeptide, a peptide, a free amino acid, a carbohydrate, afat, a mineral or mineral source, a vitamin, a supplement, an organism,a pharmaceutical, and an excipient. In some embodiments the at least onesecond component is a protein. In some embodiments the at least onesecond component is a nutritive protein. In some embodiments the atleast one second component is a free amino acid selected from essentialamino acids, non-essential amino acids, branch chain amino acids,non-standard amino acids and modified amino acids. In some embodimentsthe at least one second component is a free amino acid selected fromessential amino acids. In some embodiments the at least one secondcomponent is a free amino acid selected from branch chain amino acids.In some embodiments the at least one second component is Leu. In someembodiments the at least one second component is a lipid. In someembodiments the lipid is selected from a fat, oil, triglyceride,cholesterol, phospholipid, and fatty acid. In some embodiments the atleast one second component is selected from a mineral and a vitamin. Insome embodiments the at least one second component is a supplement. Insome embodiments the at least one second component is an organism. Insome embodiments the at least one second component is a pharmaceutical.In some embodiments the at least one second component is an excipient.In some embodiments the at least one excipient is selected from abuffering agent, a preservative, a stabilizer, a binder, a compactionagent, a lubricant, a dispersion enhancer, a disintegration agent, aflavoring agent, a sweetener, a coloring agent. In some embodiments thenutritive composition is formulated as a liquid solution, slurry,suspension, gel, paste, powder, or solid.

In another aspect this disclosure provides methods of making a nutritivecomposition of this disclosure, comprising providing a nutritive proteinaccording to this disclosure and combining the nutritive protein withthe at least one second component.

In another aspect this disclosure provides methods of maintaining orincreasing at least one of muscle mass, muscle strength, and functionalperformance in a subject, the method comprising providing to the subjecta sufficient amount of a nutritive protein of this disclosure, anutritive composition of this disclosure, or a nutritive compositionmade by a method of this disclosure. In some embodiments the subject isat least one of elderly, critically-medically ill, and suffering fromprotein-energy malnutrition. In some embodiments the nutritive proteinof this disclosure, nutritive composition of this disclosure, ornutritive composition made by a method of this disclosure is consumed bythe subject in coordination with performance of exercise. In someembodiments the nutritive protein of this disclosure, nutritivecomposition of this disclosure, or nutritive composition made by amethod of this disclosure is consumed by the subject by an oral,enteral, or parenteral route.

In another aspect this disclosure provides methods of maintaining orachieving a desirable body mass index in a subject, the methodcomprising providing to the subject a sufficient amount of a nutritiveprotein of this disclosure, a nutritive composition of this disclosure,or a nutritive composition made by a method of this disclosure. In someembodiments the subject is at least one of elderly, critically-medicallyill, and suffering from protein-energy malnutrition. In some embodimentsthe nutritive protein of this disclosure, nutritive composition of thisdisclosure, or nutritive composition made by a method of this disclosureis consumed by the subject in coordination with performance of exercise.In some embodiments the nutritive protein of this disclosure, nutritivecomposition of this disclosure, or nutritive composition made by amethod of this disclosure is consumed by the subject by an oral,enteral, or parenteral route.

In another aspect this disclosure provides methods of providing proteinto a subject with protein-energy malnutrition, the method comprisingproviding to the subject a sufficient amount of a nutritive protein ofthis disclosure, a nutritive composition of this disclosure, or anutritive composition made by a method of this disclosure. In someembodiments the subject is obese. In some embodiments the nutritiveprotein of this disclosure, nutritive composition of this disclosure, ornutritive composition made by a method of this disclosure is consumed bythe subject in coordination with performance of exercise. In someembodiments the nutritive protein of this disclosure, nutritivecomposition of this disclosure, or nutritive composition made by amethod of this disclosure is consumed by the subject by an oral,enteral, or parenteral route.

In another aspect this disclosure provides methods of increasingthermogenesis in a subject, the method comprising providing to thesubject a sufficient amount of a nutritive protein of this disclosure, anutritive composition of this disclosure, or a nutritive compositionmade by a method of this disclosure. In some embodiments the subject isobese. In some embodiments the nutritive protein of this disclosure,nutritive composition of this disclosure, or nutritive composition madeby a method of this disclosure is consumed by the subject incoordination with performance of exercise. In some embodiments thenutritive protein of this disclosure, nutritive composition of thisdisclosure, or nutritive composition made by a method of this disclosureis consumed by the subject by an oral, enteral, or parenteral route.

In another aspect this disclosure provides methods of inducing at leastone of a satiation response and a satiety response in a subject, themethod comprising providing to the subject a sufficient amount of anutritive protein of this disclosure, a nutritive composition of thisdisclosure, or a nutritive composition made by a method of thisdisclosure. In some embodiments the subject is obese. In someembodiments the nutritive protein of this disclosure, nutritivecomposition of this disclosure, or nutritive composition made by amethod of this disclosure is consumed by the subject in coordinationwith performance of exercise. In some embodiments the nutritive proteinof this disclosure, nutritive composition of this disclosure, ornutritive composition made by a method of this disclosure is consumed bythe subject by an oral, enteral, or parenteral route.

In another aspect this disclosure provides methods of treating at leastone of cachexia, sarcopenia and frailty in a subject, the methodcomprising providing to the subject a sufficient amount of a nutritiveprotein of this disclosure, a nutritive composition of this disclosure,or a nutritive composition made by a method of this disclosure. In someembodiments the nutritive protein of this disclosure, nutritivecomposition of this disclosure, or nutritive composition made by amethod of this disclosure is consumed by the subject by an oral,enteral, or parenteral route.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a two dimensional histogram indicating the relativelikelihood (on a log scale) of a protein being expressed in an E. coliexpression screen as a function of solvation score (y-axis) andaggregation score (x-axis).

FIG. 2 shows a two dimensional histogram indicating the relativelikelihood (on a log scale) of a protein being solubly expressed in anE. coli expression screen as a function of solvation score (y-axis) andaggregation score (x-axisNN

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall include theplural and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art. Certain referencesand other documents cited herein are expressly incorporated herein byreference. Additionally, all UniProt/SwissProt records cited herein arehereby incorporated herein by reference. In case of conflict, thepresent specification, including definitions, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The methods and techniques of the present disclosure are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (2001); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Taylor andDrickamer, Introduction to Glycobiology, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold,N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press(1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press(1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press(1999). Many molecular biology and genetic techniques applicable tocyanobacteria are described in Heidorn et al., “Synthetic Biology inCyanobacteria: Engineering and Analyzing Novel Functions,” Methods inEnzymology, Vol. 497, Ch. 24 (2011), which is hereby incorporated hereinby reference.

This disclosure refers to sequence database entries (e.g.,UniProt/SwissProt) for certain protein and gene sequences that arepublished on the internet, as well as other information on the internet.The skilled artisan understands that information on the internet,including sequence database entries, is updated from time to time andthat, for example, the reference number used to refer to a particularsequence can change. Where reference is made to a public database ofsequence information or other information on the internet, it isunderstood that such changes can occur and particular embodiments ofinformation on the internet can come and go. Because the skilled artisancan find equivalent information by searching on the internet, areference to an internet web page address or a sequence database entryevidences the availability and public dissemination of the informationin question.

Before the present proteins, compositions, methods, and otherembodiments are disclosed and described, it is to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. It must be notedthat, as used in the specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise.

The term “comprising” as used herein is synonymous with “including” or“containing”, and is inclusive or open-ended and does not excludeadditional, unrecited members, elements or method steps.

This disclosure makes reference to amino acids. The full name of theamino acids is used interchangeably with the standard three letter andone letter abbreviations for each. For the avoidance of doubt, thoseare: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Asparticacid (Asp, D), Cysteine (Cys, C), Glutamic Acid (Glu, E), Glutamine(Gln, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I),Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine(Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T),Tryptophan (Trp, W), Tyrosine (Tyr, Y), Valine (Val, V).

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, in a Petri dish, etc., rather than within an organism (e.g.,animal, plant, or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, or microbe).

As used herein, the term “isolated” refers to a substance or entity thathas been (1) separated from at least some of the components with whichit was associated when initially produced (whether in nature or in anexperimental setting), and/or (2) produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. As usedherein, a substance is “pure” if it is substantially free of othercomponents.

As used herein, a “branch chain amino acid” is an amino acid selectedfrom Leucine, Isoleucine, and Valine.

As used herein, an “essential amino acid” is an amino acid selected fromHistidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine,Threonine, Tryptophan, and Valine.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that typically contains less than about 50 amino acids and moretypically less than about 30 amino acids. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities. For the avoidance ofdoubt, a “polypeptide” may be any length greater two amino acids.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) exists in a purity not found in nature, wherepurity can be adjudged with respect to the presence of other cellularmaterial (e.g., is free of other proteins from the same species) (3) isexpressed by a cell from a different species, or (4) does not occur innature (e.g., it is a fragment of a polypeptide found in nature or itincludes amino acid analogs or derivatives not found in nature orlinkages other than standard peptide bonds). Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A polypeptide or protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.As thus defined, “isolated” does not necessarily require that theprotein, polypeptide, peptide or oligopeptide so described has beenphysically removed from a cell in which it was synthesized.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide, such as a naturallyoccurring protein. In an embodiment, the polypeptide fragment is acontiguous sequence in which the amino acid sequence of the fragment isidentical to the corresponding positions in the naturally-occurringsequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 aminoacids long, or at least 12, 14, 16 or 18 amino acids long, or at least20 amino acids long, or at least 25, 30, 35, 40 or 45, amino acids, orat least 50 or 60 amino acids long, or at least 70 amino acids long.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements that can be from two or moredifferent proteins. A fusion protein comprises at least 10 contiguousamino acids from a polypeptide of interest, or at least 20 or 30 aminoacids, or at least 40, 50 or 60 amino acids, or at least 75, 100 or 125amino acids. The heterologous polypeptide included within the fusionprotein is usually at least 6 amino acids in length, or at least 8 aminoacids in length, or at least 15, 20, or 25 amino acids in length.Fusions that include larger polypeptides, such as an IgG Fc region, andeven entire proteins, such as the green fluorescent protein (“GFP”)chromophore-containing proteins, have particular utility. Fusionproteins can be produced recombinantly by constructing a nucleic acidsequence which encodes the polypeptide or a fragment thereof in framewith a nucleic acid sequence encoding a different protein or peptide andthen expressing the fusion protein. Alternatively, a fusion protein canbe produced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

As used herein, a protein has “homology” or is “homologous” to a secondprotein if the nucleic acid sequence that encodes the protein has asimilar sequence to the nucleic acid sequence that encodes the secondprotein. Alternatively, a protein has homology to a second protein ifthe two proteins have similar amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89.

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine, Threonine; 2) Aspartic Acid,Glutamic Acid; 3) Asparagine, Glutamine; 4) Arginine, Lysine; 5)Isoleucine, Leucine, Methionine, Alanine, Valine, and 6) Phenylalanine,Tyrosine, Tryptophan.

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using a measure of homology assignedto various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild-type protein and amutein thereof. See, e.g., GCG Version 6.1.

An exemplary algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Exemplary parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62. The length of polypeptide sequences compared for homologywill generally be at least about 16 amino acid residues, or at leastabout 20 residues, or at least about 24 residues, or at least about 28residues, or more than about 35 residues. When searching a databasecontaining sequences from a large number of different organisms, it maybe useful to compare amino acid sequences. Database searching usingamino acid sequences can be measured by algorithms other than blastpknown in the art. For instance, polypeptide sequences can be comparedusing FASTA, a program in GCG Version 6.1. FASTA provides alignments andpercent sequence identity of the regions of the best overlap between thequery and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990).For example, percent sequence identity between amino acid sequences canbe determined using FASTA with its default parameters (a word size of 2and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereinincorporated by reference.

In some embodiments, polymeric molecules (e.g., a polypeptide sequenceor nucleic acid sequence) are considered to be “homologous” to oneanother if their sequences are at least 25%, at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical. In some embodiments,polymeric molecules are considered to be “homologous” to one another iftheir sequences are at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% similar. The term “homologous”necessarily refers to a comparison between at least two sequences(nucleotides sequences or amino acid sequences). In some embodiments,two nucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50% identical, at leastabout 60% identical, at least about 70% identical, at least about 80%identical, or at least about 90% identical for at least one stretch ofat least about 20 amino acids. In some embodiments, homologousnucleotide sequences are characterized by the ability to encode astretch of at least 4-5 uniquely specified amino acids. Both theidentity and the approximate spacing of these amino acids relative toone another must be considered for nucleotide sequences to be consideredhomologous. In some embodiments of nucleotide sequences less than 60nucleotides in length, homology is determined by the ability to encode astretch of at least 4-5 uniquely specified amino acids. In someembodiments, two protein sequences are considered to be homologous ifthe proteins are at least about 50% identical, at least about 60%identical, at least about 70% identical, at least about 80% identical,or at least about 90% identical for at least one stretch of at leastabout 20 amino acids.

As used herein, a “modified derivative” refers to polypeptides orfragments thereof that are substantially homologous in primarystructural sequence to a reference polypeptide sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thereference polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands that bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands that canserve as specific binding pair members for a labeled ligand. The choiceof label depends on the sensitivity required, ease of conjugation withthe primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002).

As used herein, “polypeptide mutant” or “mutein” refers to a polypeptidewhose sequence contains an insertion, duplication, deletion,rearrangement or substitution of one or more amino acids compared to theamino acid sequence of a reference protein or polypeptide, such as anative or wild-type protein. A mutein may have one or more amino acidpoint substitutions, in which a single amino acid at a position has beenchanged to another amino acid, one or more insertions and/or deletions,in which one or more amino acids are inserted or deleted, respectively,in the sequence of the reference protein, and/or truncations of theamino acid sequence at either or both the amino or carboxy termini. Amutein may have the same or a different biological activity compared tothe reference protein.

In some embodiments, a mutein has, for example, at least 85% overallsequence homology to its counterpart reference protein. In someembodiments, a mutein has at least 90% overall sequence homology to thewild-type protein. In other embodiments, a mutein exhibits at least 95%sequence identity, or 98%, or 99%, or 99.5% or 99.9% overall sequenceidentity.

As used herein, a “polypeptide tag for affinity purification” is anypolypeptide that has a binding partner that can be used to isolate orpurify a second protein or polypeptide sequence of interest fused to thefirst “tag” polypeptide. Several examples are well known in the art andinclude a His-6 tag, a FLAG epitope, a c-myc epitope, a Strep-TAGII, abiotin tag, a glutathione 5-transferase (GST), a chitin binding protein(CBP), a maltose binding protein (MBP), or a metal affinity tag.

As used herein, “recombinant” refers to a biomolecule, e.g., a gene orprotein, that (1) has been removed from its naturally occurringenvironment, (2) is not associated with all or a portion of apolynucleotide in which the gene is found in nature, (3) is operativelylinked to a polynucleotide which it is not linked to in nature, or (4)does not occur in nature. The term “recombinant” can be used inreference to cloned DNA isolates, chemically synthesized polynucleotideanalogs, or polynucleotide analogs that are biologically synthesized byheterologous systems, as well as proteins and/or mRNAs encoded by suchnucleic acids. Thus, for example, a protein synthesized by amicroorganism is recombinant, for example, if it is synthesized from anmRNA synthesized from a recombinant gene present in the cell.

The term “polynucleotide”, “nucleic acid molecule”, “nucleic acid”, or“nucleic acid sequence” refers to a polymeric form of nucleotides of atleast 10 bases in length. The term includes DNA molecules (e.g., cDNA orgenomic or synthetic DNA) and RNA molecules (e.g., mRNA or syntheticRNA), as well as analogs of DNA or RNA containing non-natural nucleotideanalogs, non-native internucleoside bonds, or both. The nucleic acid canbe in any topological conformation. For instance, the nucleic acid canbe single-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation.

A “synthetic” RNA, DNA or a mixed polymer is one created outside of acell, for example one synthesized chemically.

The term “nucleic acid fragment” as used herein refers to a nucleic acidsequence that has a deletion, e.g., a 5′-terminal or 3′-terminaldeletion compared to a full-length reference nucleotide sequence. In anembodiment, the nucleic acid fragment is a contiguous sequence in whichthe nucleotide sequence of the fragment is identical to thecorresponding positions in the naturally-occurring sequence. In someembodiments fragments are at least 10, 15, 20, or 25 nucleotides long,or at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or150 nucleotides long. In some embodiments a fragment of a nucleic acidsequence is a fragment of an open reading frame sequence. In someembodiments such a fragment encodes a polypeptide fragment (as definedherein) of the protein encoded by the open reading frame nucleotidesequence.

As used herein, an endogenous nucleic acid sequence in the genome of anorganism (or the encoded protein product of that sequence) is deemed“recombinant” herein if a heterologous sequence is placed adjacent tothe endogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. In this context, aheterologous sequence is a sequence that is not naturally adjacent tothe endogenous nucleic acid sequence, whether or not the heterologoussequence is itself endogenous (originating from the same host cell orprogeny thereof) or exogenous (originating from a different host cell orprogeny thereof). By way of example, a promoter sequence can besubstituted (e.g., by homologous recombination) for the native promoterof a gene in the genome of a host cell, such that this gene has analtered expression pattern. This gene would now become “recombinant”because it is separated from at least some of the sequences thatnaturally flank it.

A nucleic acid is also considered “recombinant” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “recombinant” if it contains an insertion, deletion or apoint mutation introduced artificially, e.g., by human intervention. A“recombinant nucleic acid” also includes a nucleic acid integrated intoa host cell chromosome at a heterologous site and a nucleic acidconstruct present as an episome.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence. The term “degenerate oligonucleotide” or “degenerate primer”is used to signify an oligonucleotide capable of hybridizing with targetnucleic acid sequences that are not necessarily identical in sequencebut that are homologous to one another within one or more particularsegments.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32, and even more typically at least about36 or more nucleotides. There are a number of different algorithms knownin the art which can be used to measure nucleotide sequence identity.For instance, polynucleotide sequences can be compared using FASTA, Gapor Bestfit, which are programs in Wisconsin Package Version 10.0,Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignmentsand percent sequence identity of the regions of the best overlap betweenthe query and search sequences. Pearson, Methods Enzymol. 183:63-98(1990). For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1, hereinincorporated by reference. Alternatively, sequences can be comparedusing the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 76%, 80%, 85%, or atleast about 90%, or at least about 95%, 96%, 97%, 98% or 99% of thenucleotide bases, as measured by any well-known algorithm of sequenceidentity, such as FASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (Tm) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the Tm for the specific DNA hybridunder a particular set of conditions. The Tm is the temperature at which50% of the target sequence hybridizes to a perfectly matched probe. SeeSambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page9.51, hereby incorporated by reference. For purposes herein, “stringentconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled worker that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

As used herein, an “expression control sequence” refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toencompass, at a minimum, any component whose presence is essential forexpression, and can also encompass an additional component whosepresence is advantageous, for example, leader sequences and fusionpartner sequences.

As used herein, “operatively linked” or “operably linked” expressioncontrol sequences refers to a linkage in which the expression controlsequence is contiguous with the gene of interest to control the gene ofinterest, as well as expression control sequences that act in trans orat a distance to control the gene of interest.

As used herein, a “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which generally refersto a circular double stranded DNA loop into which additional DNAsegments may be ligated, but also includes linear double-strandedmolecules such as those resulting from amplification by the polymerasechain reaction (PCR) or from treatment of a circular plasmid with arestriction enzyme. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certain vectorsare capable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply “expression vectors”).

The term “recombinant host cell” (or simply “recombinant cell” or “hostcell”), as used herein, is intended to refer to a cell into which arecombinant nucleic acid such as a recombinant vector has beenintroduced. In some instances the word “cell” is replaced by a namespecifying a type of cell. For example, a “recombinant microorganism” isa recombinant host cell that is a microorganism host cell and a“recombinant cyanobacteria” is a recombinant host cell that is acyanobacteria host cell. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “recombinanthost cell,” “recombinant cell,” and “host cell”, as used herein. Arecombinant host cell may be an isolated cell or cell line grown inculture or may be a cell which resides in a living tissue or organism.

As used herein, the term “heterotrophic” refers to an organism thatcannot fix carbon and uses organic carbon for growth.

As used herein, the term “autotrophic” refers to an organism thatproduces complex organic compounds (such as carbohydrates, fats, andproteins) from simple inorganic molecules using energy from light (byphotosynthesis) or inorganic chemical reactions (chemosynthesis).

As used herein, “muscle mass” refers to the weight of muscle in asubject's body. Muscle mass includes the skeletal muscles, smoothmuscles (such as cardiac and digestive muscles) and the water containedin these muscles. Muscle mass of specific muscles can be determinedusing dual energy x-ray absorptiometry (DEXA) (Padden-Jones et al.,2004). Total lean body mass (minus the fat), total body mass, and bonemineral content can be measured by DEXA as well. In some embodiments achange in the muscle mass of a specific muscle of a subject isdetermined, for example by DEXA, and the change is used as a proxy forthe total change in muscle mass of the subject. Thus, for example, if asubject consumes a nutritive protein as disclosed herein and experiencesan increase over a period of time in muscle mass in a particular muscleor muscle group, it can be concluded that the subject has experienced anincrease in muscle mass.

As used herein, a “muscle strength” refers to the amount of force amuscle can produce with a single maximal effort. There are two types ofmuscle strength, static strength and dynamic strength. Static strengthrefers to isometric contraction of a muscle, where a muscle generatesforce while the muscle length remains constant and/or when there is nomovement in a joint. Examples include holding or carrying an object, orpushing against a wall. Dynamic strength refers to a muscle generatingforce that results in movement. Dynamic strength can be isotoniccontraction, where the muscle shortens under a constant load orisokinetic contraction, where the muscle contracts and shortens at aconstant speed. Dynamic strength can also include isoinertial strength.

Unless specified, “muscle strength” refers to maximum dynamic musclestrength. Maximum strength is referred to as “one repetition maximum”(1RM). This is a measurement of the greatest load (in kilograms) thatcan be fully moved (lifted, pushed or pulled) once without failure orinjury. This value can be measured directly, but doing so requires thatthe weight is increased until the subject fails to carry out theactivity to completion. Alternatively, 1RM is estimated by counting themaximum number of exercise repetitions a subject can make using a loadthat is less than the maximum amount the subject can move. Leg extensionand leg flexion are often measured in clinical trials (Borsheim et al.,“Effect of amino acid supplementation on muscle mass, strength andphysical function in elderly,” Clin Nutr 2008; 27:189-195; Paddon-Jones,et al., “Essential amino acid and carbohydrate supplementationameliorates muscle protein loss in humans during 28 days bed rest,” JClin Endocrinol Metab 2004; 89:4351-4358).

As used herein, “functional performance” refers to a functional testthat simulates daily activities. “Functional performance” is measured byany suitable accepted test, including timed-step test (step up and downfrom a 4 inch bench as fast as possible 5 times), timed floor transfertest (go from a standing position to a supine position on the floor andthereafter up to a standing position again as fast as possible for onerepetition), and physical performance battery test (static balance test,chair test, and a walking test) (Borsheim et al., “Effect of amino acidsupplementation on muscle mass, strength and physical function inelderly,” Clin Nutr 2008; 27:189-195).

As used herein, a “body mass index” or “BMI” or “Quetelet index” is asubject's weight in kilograms divided by the square of the subject'sheight in meters (kg/m²).

For adults, a frequent use of the BMI is to assess how much anindividual's body weight departs from what is normal or desirable for aperson of his or her height. The weight excess or deficiency may, inpart, be accounted for by body fat, although other factors such asmuscularity also affect BMI significantly. The World Health Organizationregards a BMI of less than 18.5 as underweight and may indicatemalnutrition, an eating disorder, or other health problems, while a BMIgreater than 25 is considered overweight and above 30 is consideredobese. (World Health Organization. BMI classification. Accessed Mar. 19,2012 http://apps.who.int/bmi/index.jsp?introPage=intro_3.html.) As usedherein a “desirable body mass index” is a body mass index of from about18.5 to about 25. Thus, if a subject has a BMI below about 18.5, then anincrease in the subject's BMI is an increase in the desirability of thesubject's BMI. If instead a subject has a BMI above about 25, then adecrease in the subject's BMI is an increase in the desirability of thesubject's BMI.

As used herein, an “elderly” mammal is one who experiences age relatedchanges in at least one of body mass index and muscle mass (e.g., agerelated sarcopenia). In some embodiments an “elderly” human is at least50 years old, at least 60 years old, at least 65 years old, at least 70years old, at least 75 years old, at least 80 years old, at least 85years old, at least 90 years old, at least 95 years old, or at least 100years old. In some embodiments and an elderly animal, mammal, or humanis a human who has experienced a loss of muscle mass from peak lifetimemuscle mass of at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, or at least 60%. Because age related changes toat least one of body mass index and muscle mass are known to correlatewith increasing age, in some embodiments an elderly mammal is identifiedor defined simply on the basis of age. Thus, in some embodiments an“elderly” human is identified or defined simply by the fact that theirage is at least 60 years old, at least 65 years old, at least 70 yearsold, at least 75 years old, at least 80 years old, at least 85 yearsold, at least 90 years old, at least 95 years old, or at least 100 yearsold, and without recourse to a measurement of at least one of body massindex and muscle mass.

As used herein, a patient is “critically-medically ill” if the patient,because of medical illness, experiences changes in at least one of bodymass index and muscle mass (e.g., sarcopenia). In some embodiments thepatient is confined to bed for at least 25%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, or 100% of theirwaking time. In some embodiments the patient is unconscious. In someembodiments the patient has been confined to bed as described in thisparagraph for at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 2weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks or longer.

As used herein, “protein-energy malnutrition” refers to a form ofmalnutrition where there is inadequate protein intake. Types includeKwashiorkor (protein malnutrition predominant), Marasmus (deficiency inboth calorie and protein nutrition), and Marasmic Kwashiorkor (markedprotein deficiency and marked calorie insufficiency signs present,sometimes referred to as the most severe form of malnutrition).

As used herein, “cachexia” refers to a multifaceted clinical syndromethat results in wasting and weight loss. It is a complex condition whereprotein catabolism exceeds protein anabolism, which makes muscle wastinga primary feature of the condition. In addition to the metabolicderangements in protein metabolism, it is also characterized by anorexiaand inflammation. These derangements plus impaired protein metabolismare responsive to nutrition therapy to varying degrees.

As used herein, “thermogenesis” is the process of heat production in amammal. Thermogenesis is accompanied by an increase in energyexpenditure. Thermogenesis is specifically the energy burned followingthe metabolism of a food component (such as protein). This may also bereferred to as the thermic effect of food. Total energy expenditure byan individual equals the sum of resting energy expenditure (energyconsumed at rest in a fasting state to support basal metabolism), thethermic effect of food, and energy expenditure related to physicalactivity. Resting energy expenditure accounts for about 65-75% of totalenergy expenditure in humans. The amount and activity of muscle mass isone influencer of resting energy expenditure. Adequate proteinconsumption to support muscle also influences resting energyexpenditure. The ingestion of protein tends to increase energyexpenditure following a meal; this is the thermic effect of food. Thethermic effect of food accounts for about 10% of total energyexpenditure in humans. While this is a small proportion of total energyexpenditure, small increases in this value can impact body weight.Protein has a higher thermic effect than fat or carbohydrate; thiseffect along with other metabolic influences of protein make it a usefulsubstrate for weight control, diabetes management and other conditions.

As used herein, “satiation” is the act of becoming full while eating ora reduced desire to eat. This halts or diminishes eating.

As used herein, “satiety” is the act of remaining full after a mealwhich manifests as the period of no eating follow the meal.

As used herein, “exercise” is, most broadly, any bodily activity thatenhances or maintains physical fitness and overall health and wellness.Exercise is performed for various reasons including strengtheningmuscles and the cardiovascular system, honing athletic skills, weightloss or maintenance, as well as for the purpose of enjoyment.

As used herein, a “sufficient amount” is an amount of a protein orpolypeptide disclosed herein that is sufficient to cause a desiredeffect. For example, if an increase in muscle mass is desired, asufficient amount is an amount that causes an increase in muscle mass ina subject over a period of time. A sufficient amount of a protein orpolypeptide fragment can be provided directly, i.e., by administeringthe protein or polypeptide fragment to a subject, or it can be providedas part of a composition comprising the protein or polypeptide fragment.Modes of administration are discussed elsewhere herein.

As used herein, the term “mammal” refers to any member of the taxonomicclass mammalia, including placental mammals and marsupial mammals. Thus,“mammal” includes humans, primates, livestock, and laboratory mammals.Exemplary mammals include a rodent, a mouse, a rat, a rabbit, a dog, acat, a sheep, a horse, a goat, a llama, cattle, a primate, a pig, andany other mammal. In some embodiments, the mammal is at least one of atransgenic mammal, a genetically-engineered mammal, and a cloned mammal.

A. Nutritive Proteins

For the purposes of this disclosure, a “nutritive protein” is a proteinthat contains a desirable amount of essential amino acids. In someembodiments, the nutritive protein comprises at least 30% essentialamino acids by weight. In some embodiments, the nutritive proteincomprises at least 40% essential amino acids by weight. In someembodiments, the nutritive protein comprises at least 50% essentialamino acids by weight. In some embodiments the nutritive proteincomprises or consists of a protein or fragment of a protein thatnaturally occurs in an edible species. In its broadest sense, an “ediblespecies” encompasses any species known to be eaten without deleteriouseffect by at least one type of mammal. A deleterious effect includes apoisonous effect and a toxic effect. In some embodiments an ediblespecies is a species known to be eaten by humans without deleteriouseffect. Some edible species are an infrequent but known component of thediet of only a small group of a type of mammal in a limited geographiclocation while others are a dietary staple throughout much of the world.In other embodiments an edible species is one not known to be previouslyeaten by any mammal, but that is demonstrated to be edible upon testing.Edible species include but are not limited to Gossypium turneri,Pleurotus cornucopiae, Glycine max, Oryza sativa, Thunnus obesus, Abiesbracteata, Acomys ignitus, Lathyrus aphaca, Bos gaurus, Raphicerusmelanotis, Phoca groenlandica, Acipenser sinensis, Viverra tangalunga,Pleurotus sajor-caju, Fagopyrum tataricum, Pinus strobus, Ipomoea nil,Taxus cuspidata, Ipomoea wrightii, Mya arenaria, Actinidia deliciosa,Gazella granti, Populus tremula, Prunus domestica, Larus argentatus,Vicia villosa, Sargocentron punctatissimum, Silene latifolia,Lagenodelphis hosei, Spisula solidissima, Crossarchus obscurus,Phaseolus angularis, Lathyrus vestitus, Oncorhynchus gorbuscha,Alligator mississippiensis, Pinus halepensis, Larus canus, Brassicanapus, Silene cucubalus, Phoca fasciata, Gazella bennettii, Pinus taeda,Taxus canadensis, Zamiafurfuracea, Pinus yunnanensis, Pinus wallichiana,Asparagus officinalis, Capsicum baccatum, Pinus longaeva, Taxus baccata,Pinus sibirica, Citrus sinensis, Sargocentron xantherythrum, Bisonbison, Gazella thomsonii, Vicia sativa, Branta canadensis, Apiumgraveolens, Acer campestre, Coriandrum sativum, Silene conica, Lactucasativa, Capsicum chinense, Abies veitchii, Capra hircus, Gazella spekei,Oncorhynchus keta, Ipomoea obscura, Cucumis melo var. conomon, Phocahispida, Vulpes vulpes, Ipomoea quamoclit, Solanum habrochaites, Populussp., Pinus rigida, Quercus lyrata, Phaseolus coccineus, Larusridibundus, Sargocentron spiniferum, Thunnus thynnus, Vulpes lagopus,Bos gaurusfrontalis, Acer opalus, Acer palmatum, Quercus ilex, Pinusmugo, Grus antigone, Pinus uncinata, Prunus mume, Oncorhynchustschawytscha, Gazella subgutturosa, Vulpes zerda, Pinus coulteri,Gossypium barbadense, Acer pseudoplatanus, Oncorhynchus nerka, Susbarbatus, Fagopyrum esculentum subsp. Ancestrale, Cynara cardunculus,Phaseolus aureus, Populus nigra, Gossypium schwendimanii, Solanumchacoense, Quercus rubra, Cucumis sativus, Equus burchelli, Oncorhynchuskisutch, Pinus radiata, Phoca vitulina richardsi, Grus nigricollis,Abies grandis, Oncorhynchus masou, Spinacia oleracea, Solanum chilense,Addax nasomaculatus, Ipomoea batatas, Equus grevyi, Abies sachalinensis,Pinus pinea, Hipposideros commersoni, Crocus nudiflorus, Citrus maxima,Acipenser transmontanus, Gossypium gossypioides, Viverra zibetha,Quercus cerris, Anser indicus, Pinus balfouriana, Silene otites,Oncorhynchus sp., Viverra megaspila, Bos mutus grunniens, Pinuselliottii, Equus hemionus kulan, Capra ibex ibex, Allium sativum,Raphanus sativus, Pinus echinata, Prunus serotina, Sargocentron diadema,Silene gallica, Brassica oleracea, Daucus carota, Oncorhynchus mykiss,Brassica oleracea var. alboglabra, Gossypium hirsutum, Abies alba,Citrus reticulata, Cichorium intybus, Bos sauveli, Lama glama, Zea mays,Acorus gramineus, Vulpes macrotis, Ovis ammon darwini, Raphicerussharpei, Pinus contorta, Bos indicus, Capra sibirica, Pinus ponderosa,Prunus dulcis, Solanum sogarandinum, Ipomoea aquatica, Lagenorhynchusalbirostris, Ovis canadensis, Prunus avium, Gazella dama, Thunnusalalunga, Silene pratensis, Pinus cembra, Crocus sativus, Citrulluslanatus, Gazella rufifrons, Brassica tournefortii, Capra falconeri,Bubalus mindorensis, Pinus palustris, Prunus laurocerasus, Grus vipio,Ipomoea purpurea, Pinus leiophylla, Lagenorhynchus obscurus, Raphiceruscampestris, Brassica rapa subsp. Pekinensis, Acmella radicans, Ipomoeatriloba, Pinus patula, Cucumis melo, Pinus virginiana, Solanumlycopersicum, Pinus densiflora, Pinus engelmannii, Quercus robur,Ipomoea setosa, Pleurotus djamor, Hipposideros diadema, Ovis aries,Sargocentron microstoma, Brassica oleracea var. italica, Capracylindricornis, Populus kitakamiensis, Allium textile, Vicia faba,Fagopyrum esculentum, Bison priscus, Quercus suber, Lagophyllaramosissima, Acrantophis madagascariensis, Acipenser baerii, Capsicumannuum, Triticum aestivum, Xenopus laevis, Phoca sibirica, Acipensernaccarii, Actinidia chinensis, Ovis dalli, Solanum tuberosum, Bubaluscarabanensis, Citrus jambhiri, Bison bonasus, Equus asinus, Bubalusdepressicornis, Pleurotus eryngii, Solanum demissum, Ovis vignei, Zeamays subsp. Parviglumis, Lathyrus tingitanus, Welwitschia mirabilis,Grus rubicunda, Ipomoea coccinea, Allium cepa, Gazella soemmerringii,Brassica rapa, Lama vicugna, Solanum peruvianum, Xenopus borealis, Capracaucasica, Thunnus albacares, Equus zebra, Gallus gallus, Solanumbulbocastanum, Hipposideros terasensis, Lagenorhynchus acutus,Hippopotamus amphibius, Pinus koraiensis, Acer monspessulanum, Populusdeltoides, Populus trichocarpa, Acipenser guldenstadti, Pinusthunbergii, Brassica oleracea var. capitata, Abyssocottus korotneffi,Gazella cuvieri, Abies homolepis, Abies holophylla, Gazella gazella,Pinus parviflora, Brassica oleracea var. acephala, Cucurbita pepo, Pinusarmandii, Abies mariesii, Thunnus thynnus orientalis, Citrus unshiu,Solanum cheesmanii, Lagenorhynchus obliquidens, Acer platanoides, Citruslimon, Acrantophis dumerili, Solanum commersonii, Gossypium arboreum,Prunus persica, Pleurotus ostreatus, Abies firma, Gazella leptoceros,Salmo salar, Homarus americanus, Abies magnifica, Bos javanicus, Phocalargha, Sus cebifrons, Solanum melongena, Phoca vitulina, Pinussylvestris, Zamia floridana, Vulpes corsac, Allium porrum, Phocacaspica, Vulpes chama, Taxus chinensis, Brassica oleracea var. botrytis,Anser anser anser, Phaseolus lunatus, Brassica campestris, Acersaccharum, Pinus pumila, Solanum pennellii, Pinus edulis, Ipomoeacordatotriloba, Populus alba, Oncorhynchus clarki, Quercus petraea, Susverrucosus, Equus caballus przewalskii, Populus euphratica, Xenopustropicalis, Taxus brevifolia, Lama guanicoe, Pinus banksiana, Solanumnigrum, Sus celebensis, Brassica juncea, Lagenorhynchus cruciger,Populus tremuloides, Pinus pungens, Bubalus quarlesi, Quercusgamelliflora, Ovis orientalis musimon, Bubalus bubalis, Pinusluchuensis, Sus philippensis, Phaseolus vulgaris, Salmo trutta,Acipenser persicus, Solanum brevidens, Pinus resinosa, Hippotragusniger, Capra nubiana, Asparagus scaber, Ipomoea platensis, Sus scrofa,Capra aegagrus, Lathyrus sativus, Sargocentron tiere, Hippoglossushippoglossus, Acorus americanus, Equus caballus, Bos taurus, Barbareavulgaris, Lama guanicoe pacos, Pinus pinaster, Octopus vulgaris, Solanumcrispum, Hippotragus equinus, Equus burchellii antiquorum, Crossarchusalexandri, Ipomoea alba, Triticum monococcum, Populus jackii,Lagenorhynchus australis, Gazella dorcas, Quercus coccifera, Ansercaerulescens, Acorus calamus, Pinus roxburghii, Pinus tabuliformis,Zamia fischeri, Grus carunculatus, Acomys cahirinus, Cucumis melo var.reticulatus, Gallus lafayettei, Pisum sativum, Pinus attenuata, Pinusclausa, Gazella saudiya, Capra ibex, Ipomoea trifida, Zea luxurians,Pinus krempfii, Acomys wilsoni, Petroselinum crispum, Quercus palustris,Triticum timopheevi, Meleagris gallopavo, Brassica oleracea, Brassicaoleracea, Beta vulgaris, Solanum lycopersicum, Phaseolus vulgaris,Xiphias gladius, Morone saxatilis, Micropterus salmoides, Placopectenmagellanicus, Sprattus sprattus, Clupea harengus, Engraulisencrasicolus, Cucurbita maxima, Agaricus bisporus, Musaacuminata×balbisiana, Malus domestica, Cicer arietinum, Anasplatyrhynchos, Vaccinium macrocarpum, Rubus idaeus×strigosus, Vacciniumangustifolium, Fragaria ananassa, Rubusfruticosus, Cucumis melo, Ananascomosus, Cucurbita pepo, Cucurbita moschata, Sus scrofa domesticus,Ocimum basilicum, Rosmarinus officinalis, Foeniculum vulgare, Rheumrhabarbarum, Carica papaya, Mangifera indica, Actinidia deliciosa,Prunus armeniaca, Prunus avium, Cocos nucifera, Olea europaea, Pyruscommunis, Ficus carica, Passiflora edulis, Oryza sativa subsp. Japonica,Oryza sativa subsp. Indica, Coturnix coturnix, Saccharomyces cerevisiae.

In some embodiments the nutritive protein comprises or consists of aderivative or mutein of a protein or fragment of a protein thatnaturally occurs in an edible species. Such a nutritive protein may bereferred to as an “engineered nutritive protein.” In such embodimentsthe natural protein or fragment thereof is a “reference” protein orpolypeptide and the engineered nutritive protein or a first polypeptidesequence thereof comprises at least one sequence modification relativeto the amino acid sequence of the reference protein or polypeptide. Forexample, in some embodiments the engineered nutritive protein or firstpolypeptide sequence thereof is at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 99.5% homologous to at least one referencenutritive protein amino acid sequence. Typically the ratio of at leastone of branch chain amino acid residues to total amino acid residues,essential amino acid residues to total amino acid residues, and leucineresidues to total amino acid residues, present in the engineerednutritive protein or a first polypeptide sequence thereof is greaterthan the corresponding ratio of at least one of branch chain amino acidresidues to total amino acid residues, essential amino acid residues tototal amino acid residues, and leucine residues to total amino acidresidues present in the reference nutritive protein or polypeptidesequence.

In some embodiments the nutritive protein is an abundant protein in foodor a derivative or mutein thereof, or is a fragment of an abundantprotein in food or a derivative or mutein thereof. An abundant proteinis a protein that is present in a higher concentration in a foodrelative to other proteins present in the food. The food can be a knowncomponent of the diet of only a small group of a type of mammal in alimited geographic location, or a dietary staple throughout much of theworld. In some embodiments the abundant protein in food is selected fromchicken egg proteins such as ovalbumin, ovotransferrin, and ovomucuoid;meat proteins such as myosin, actin, tropomyosin, collagen, andtroponin; cereal proteins such as casein, alpha1 casein, alpha2 casein,beta casein, kappa casein, beta-lactoglobulin, alpha-lactalbumin,glycinin, beta-conglycinin, glutelin, prolamine, gliadin, glutenin,albumin, globulin; chicken muscle proteins such as albumin, enolase,creatine kinase, phosphoglycerate mutase, triosephosphate isomerase,apolipoprotein, ovotransferrin, phosphoglucomutase, phosphoglyceratekinase, glycerol-3-phosphate dehydrogenase, glyceraldehyde 3-phosphatedehydrogenase, hemoglobin, cofilin, glycogen phosphorylase,fructose-1,6-bisphosphatase, actin, myosin, tropomyosin a-chain, caseinkinase, glycogen phosphorylase, fructose-1,6-bisphosphatase, aldolase,tubulin, vimentin, endoplasmin, lactate dehydrogenase, destrin,transthyretin, fructose bisphosphate aldolase, carbonic anhydrase,aldehyde dehydrogenase, annexin, adenosyl homocysteinase; pork muscleproteins such as actin, myosin, enolase, titin, cofilin,phosphoglycerate kinase, enolase, pyruvate dehydrogenase, glycogenphosphorylase, triosephosphate isomerase, myokinase; and fish proteinssuch as parvalbumin, pyruvate dehydrogenase, desmin, and triosephosphateisomerase.

Three natural sources of protein generally regarded as good sources ofhigh quality amino acids are whey protein, egg protein, and soy protein.Each source comprises multiple proteins. Table 1 presents the weightproportional representation of each amino acid in the protein source (gAA/g protein) expressed as a percentage.

TABLE 1 Amino Acid Whey Egg Soy Isoleucine 6.5% 5.5% 5.0% Leucine 11.0% 8.6% 8.0% Lysine 9.1% 7.2% 6.3% Methionine 2.1% 3.1% 1.3% Phenylalanine3.4% 5.3% 1.2% Threonine 7.0% 4.8% 3.7% Tryptophan 1.7% 1.2% 1.3% Valine6.2% 6.1% 4.9% Histidine 2.0% 2.4% 2.7% Other 51.7%  49.5%  60.4% 

Table 2 presents the weight proportion of each protein source that isessential amino acids, branched chain amino acids (L, I, and V), andleucine (L) (alone).

TABLE 2 Protein Essential Branch Chain Leucine Source Amino Acids AminoAcids Whey 49.0% 23.7% 11.0%  Egg 50.5% 20.1% 8.6% Soy 39.6% 17.9% 8.0%

The sources relied on to determine the amino acid content of Whey are:Belitz H D., Grosch W., and Schieberle P. Food Chemistry (4th Ed).Springer-Verlag, Berlin Heidelberg 2009;http://www.gnc.com/product/index.jsp?productId=2986027;http://www.nutrabio.com/Products/whey_protein_concentrate.htm; andhttp://nutrabio.com/Products/whey_protein_isolate.htm. The amino acidcontent values from those sources were averaged to give the numberspresented in Tables 1 and 2. The source for soy protein is Egg, NationalNutrient Database for Standard Reference, Release 24(http://ndb.nal.usda.gov/ndb/foods/list). The source for soy protein isSelf Nutrition Data(http://nutritiondata.self.com/facts/legumes-and-legume-products/4389/2).

As used herein, “whey protein” or “whey” means a protein mixturecomprising an amino acid composition according to Tables 1 and 2. Asused herein, whey protein comprises 49% essential amino acids, 24%branch chain amino acids, and 11% leucine, by weight.

As used herein, “egg protein” or “egg” means a protein mixturecomprising an amino acid composition according to Tables 1 and 2. Asused herein, egg protein comprises 51% essential amino acids, 20% branchchain amino acids, and 9% leucine, by weight.

As used herein, “soy protein” or “soy” means a protein mixturecomprising an amino acid composition according to Tables 1 and 2. Asused herein, soy protein comprises 40% essential amino acids, 18% branchchain amino acids, and 8% leucine, by weight.

In some instances herein the portion of amino acid(s) of a particulartype within a polypeptide, protein or a composition is quantified basedon the weight ratio of the type of amino acid(s) to the total weight ofamino acids present in the polypeptide, protein or composition inquestion. This value is calculated by dividing the weight of theparticular amino acid(s) in the polypeptide, protein or a composition bythe weight of all amino acids present in the polypeptide, protein or acomposition.

In other instances the ratio of a particular type of amino acid(s)residues present in a polypeptide or protein to the total number ofamino acids present in the polypeptide or protein in question is used.This value is calculated by dividing the number of the amino acid(s) inquestion that is present in each molecule of the polypeptide or proteinby the total number of amino acid residues present in each molecule ofthe polypeptide or protein. A skilled artisan appreciates that these twomethods are interchangeable and that the weight proportion of a type ofamino acid(s) present in a polypeptide or protein can be converted to aratio of the particular type of amino acid residue(s), and vice versa.

In certain embodiments herein the weight proportion of branched chainamino acids, leucine, and/or essential amino acids in whey, egg, or soyis used as a benchmark to measure the amino acid composition of apolypeptide, a protein, or a composition comprising at least one of apolypeptide and a protein. In those embodiments it is understood thatthe two measures are not completely equivalent, but it is alsounderstood that the measures result in measurements that are similarenough to use for this purpose. For example, when a protein of interestis characterized as comprising a ratio of branch chain amino acidresidues to total amino acid residues that is equal to or greater than24% (the weight proportion of branch chain amino acid residues presentin whey), that is a precise description of the branch chain amino acidcontent of the protein. At the same time, the weight proportion ofbranch chain amino acid residues present in that protein is notnecessarily exactly equal to 24%. Even so, the skilled artisanunderstands that this is a useful comparison. If provided with the totalnumber of amino acid residues present in the protein of interest theskilled artisan can also determine the weight proportion of branch chainamino acid residues in the protein of interest.

In some embodiments herein a fragment of a naturally-occurring nutritiveprotein is selected and optionally isolated. In some embodiments thefragment comprises at least 25 amino acids. In some embodiments thefragment comprises at least 50 amino acids. In some embodiments thefragment consists of at least 25 amino acids. In some embodiments thefragment consists of at least 50 amino acids. In some embodiments anisolated recombinant nutritive protein is provided. In some embodimentsthe nutritive protein comprises a first polypeptide sequence, and thefirst polypeptide sequence comprises a fragment of at least 25 or atleast 50 amino acids of a naturally-occurring nutritive protein. In someembodiments the nutritive proteins is isolated. In some embodiments thenutritive proteins are recombinant. In some embodiments the nutritiveproteins comprise a first polypeptide sequence comprising a fragment ofat least 50 amino acids of a naturally-occurring nutritive protein. Insome embodiments the nutritive proteins are isolated recombinantnutritive proteins. In some embodiments the isolated recombinantnutritive proteins disclosed herein are provided in a non-isolatedand/or non-recombinant form

In some embodiments a nutritive protein according to this disclosurecomprises a first polypeptide sequence comprising a fragment of anaturally-occurring nutritive protein. In some embodiments of thenutritive protein, the nutritive protein consists of the firstpolypeptide sequence. In some embodiments of the nutritive protein, thenutritive protein consists of the fragment of a naturally-occurringnutritive protein.

In some embodiments a nutritive protein according to this disclosurecomprises a first polypeptide sequence that comprises ratio of branchchain amino acid residues to total amino acid residues that is equal toor greater than the ratio of branch chain amino acid residues to totalamino acid residues present in at least one of whey protein, eggprotein, and soy protein. Thus, in such embodiments the nutritiveprotein comprises a first polypeptide sequence that comprises a ratio ofbranch chain amino acid residues to total amino acid residues that isequal to or greater than a ratio selected from 24%, 20%, and 18%.

In some embodiments a nutritive protein according to this disclosurecomprises a first polypeptide sequence that comprises a ratio of Lresidues to total amino acid residues that is equal to or greater thanthe ratio of L residues to total amino acid residues present in at leastone of whey protein, egg protein, and soy protein. Thus, in suchembodiments the nutritive protein comprises a first polypeptide sequencethat comprises a ratio of L residues to total amino acid residues thatis equal to or greater than a ratio selected from 11%, 9%, and 8%.

In some embodiments a nutritive protein according to this disclosurecomprises a first polypeptide sequence that comprises a ratio ofessential amino acid residues to total amino acid residues that is equalto or greater than the ratio of essential amino acid residues to totalamino acid residues present in at least one of whey protein, eggprotein, and soy protein. Thus, in such embodiments the nutritiveprotein comprises a first polypeptide sequence that comprises a ratio ofessential amino acid residues to total amino acid residues that is equalto or greater than a ratio selected from 49%, 51%, and 40%.

In some embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of branch chain amino acid residues tototal amino acid residues that is equal to or greater than the ratio ofbranch chain amino acid residues to total amino acid residues present inat least one of whey protein, egg protein, and soy protein; andcomprises a first polypeptide sequence that comprises a ratio of Lresidues to total amino acid residues that is equal to or greater thanthe ratio of L residues to total amino acid residues present in at leastone of whey protein, egg protein, and soy protein. In some suchembodiments the nutritive protein further comprises a first polypeptidesequence that comprises a ratio of essential amino acid residues tototal amino acid residues that is equal to or greater than the ratio ofessential amino acid residues to total amino acid residues present in atleast one of whey protein, egg protein, and soy protein.

In some embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of branch chain amino acid residues tototal amino acid residues equal to or greater than 24% and a ratio of Lresidues to total amino acid residues that is equal to or greater than11%. In some such embodiments the nutritive protein further comprises afirst polypeptide sequence that comprises a ratio of essential aminoacid residues to total amino acid residues equal to or greater than 49%.

In some embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of branch chain amino acid residues tototal amino acid residues equal to or greater than 20% and a ratio of Lresidues to total amino acid residues that is equal to or greater than9%. In some such embodiments the nutritive protein further comprises afirst polypeptide sequence that comprises a ratio of essential aminoacid residues to total amino acid residues equal to or greater than 51%.

In some embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of branch chain amino acid residues tototal amino acid residues equal to or greater than 18% and a ratio of Lresidues to total amino acid residues that is equal to or greater than8%. In some such embodiments the nutritive protein further comprises afirst polypeptide sequence that comprises a ratio of essential aminoacid residues to total amino acid residues equal to or greater than 40%.

In some embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of branch chain amino acid residues tototal amino acid residues that is equal to or greater than the ratio ofbranch chain amino acid residues to total amino acid residues present inat least one of whey protein, egg protein, and soy protein; andcomprises a first polypeptide sequence that comprises a ratio ofessential amino acid residues to total amino acid residues that is equalto or greater than the ratio of essential amino acid residues to totalamino acid residues present in at least one of whey protein, eggprotein, and soy protein. In some embodiments the nutritive proteincomprises a first polypeptide sequence that comprises a ratio of branchchain amino acid residues to total amino acid residues equal to orgreater than 24% and a ratio of essential amino acid residues to totalamino acid residues that is equal to or greater than 49%. In someembodiments the nutritive protein comprises a first polypeptide sequencethat comprises a ratio of branch chain amino acid residues to totalamino acid residues equal to or greater than 20% and a ratio ofessential amino acid residues to total amino acid residues that is equalto or greater than 51%. In some embodiments the nutritive proteincomprises a first polypeptide sequence that comprises a ratio of branchchain amino acid residues to total amino acid residues equal to orgreater than 18% and a ratio of essential amino acid residues to totalamino acid residues that is equal to or greater than 40%.

In some embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of L residues to total amino acidresidues that is equal to or greater than the ratio of L residues tototal amino acid residues present in at least one of whey protein, eggprotein, and soy protein; and comprises a first polypeptide sequencethat comprises a ratio of essential amino acid residues to total aminoacid residues that is equal to or greater than the ratio of essentialamino acid residues to total amino acid residues present in at least oneof whey protein, egg protein, and soy protein. In some embodiments thenutritive protein comprises a first polypeptide sequence that comprisesa ratio of L residues to total amino acid residues equal to or greaterthan 11% and a ratio of essential amino acid residues to total aminoacid residues that is equal to or greater than 49%. In some embodimentsthe nutritive protein comprises a first polypeptide sequence thatcomprises a ratio of L amino acid residues to total amino acid residuesequal to or greater than 9% and a ratio of essential amino acid residuesto total amino acid residues that is equal to or greater than 51%. Insome embodiments the nutritive protein comprises a first polypeptidesequence that comprises a ratio of L amino acid residues to total aminoacid residues equal to or greater than 8% and a ratio of essential aminoacid residues to total amino acid residues that is equal to or greaterthan 40%.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24%, a ratio of L residues to total amino acid residuesthat is equal to or greater than 11%, a ratio of essential amino acidresidues to total amino acid residues equal to or greater than 49%, andcomprises at least one of every essential amino acid, and the firstpolypeptide sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 1000.In some embodiments the first polypeptide sequence is selected from amodified derivative of SEQ ID NO: 1 to SEQ ID NO: 1000. In someembodiments the first polypeptide sequence is selected from a mutein ofSEQ ID NO: 1 to SEQ ID NO: 1000.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24%, a ratio of L residues to total amino acid residuesthat is equal to or greater than 11%, and a ratio of essential aminoacid residues to total amino acid residues equal to or greater than 49%,and the first polypeptide sequence is selected from SEQ ID NO: 1001 toSEQ ID NO: 1414. In some embodiments the first polypeptide sequence isselected from a modified derivative of SEQ ID NO: 1001 to SEQ ID NO:1414. In some embodiments the first polypeptide sequence is selectedfrom a mutein of SEQ ID NO: 1001 to SEQ ID NO: 1414.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24%, a ratio of essential amino acid residues to totalamino acid residues equal to or greater than 49%, and comprises at leastone of every essential amino acid, and the first polypeptide sequence isselected from SEQ ID NO: 1415 to SEQ ID NO: 1899. In some embodimentsthe first polypeptide sequence is selected from a modified derivative ofSEQ ID NO: 1415 to SEQ ID NO: 1899. In some embodiments the firstpolypeptide sequence is selected from a mutein of SEQ ID NO: 1415 to SEQID NO: 1899.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofessential amino acid residues to total amino acid residues equal to orgreater than 49% and a ratio of L residues to total amino acid residuesthat is equal to or greater than 11%, and comprises at least one ofevery essential amino acid, and the first polypeptide sequence isselected from SEQ ID NO: 1900 to SEQ ID NO: 2102. In some embodimentsthe first polypeptide sequence is selected from a modified derivative ofSEQ ID NO: 1900 to SEQ ID NO: 2102. In some embodiments the firstpolypeptide sequence is selected from a mutein of SEQ ID NO: 1900 to SEQID NO: 2102.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24% and a ratio of L residues to total amino acidresidues that is equal to or greater than 11%, and comprises at leastone of every essential amino acid, and the first polypeptide sequence isselected from SEQ ID NO: 2103 to SEQ ID NO: 2518. In some embodimentsthe first polypeptide sequence is selected from a modified derivative ofSEQ ID NO: 2103 to SEQ ID NO: 2518. In some embodiments the firstpolypeptide sequence is selected from a mutein of SEQ ID NO: 2103 to SEQID NO: 2518.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24%, a ratio of L residues to total amino acid residuesthat is equal to or greater than 11%, and comprises at least one ofevery essential amino acid.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24% and a ratio of essential amino acid residues tototal amino acid residues equal to or greater than 49%.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio of Lresidues to total amino acid residues that is equal to or greater than11% and a ratio of essential amino acid residues to total amino acidresidues equal to or greater than 49%.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofessential amino acid residues to total amino acid residues equal to orgreater than 49% and comprises at least one of every essential aminoacid.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio ofbranch chain amino acid residues to total amino acid residues equal toor greater than 24% and comprises at least one of every essential aminoacid.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio of Lresidues to total amino acid residues that is equal to or greater than11% and comprises at least one of every essential amino acid.

In some embodiments of the nutritive protein, the first polypeptidesequence comprises a first polypeptide sequence comprising a ratio of Lresidues to total amino acid residues that is equal to or greater than11%.

In some embodiments of the nutritive protein, the nutritive proteinfurther comprises a second polypeptide sequence. In some embodiments thesecond polypeptide sequence consists of from 3 to 10, 5 to 20, 10 to 30,20 to 50, 25 to 75, 50 to 100 or 100 to 200 amino acids. In someembodiments the second polypeptide sequence is not derived from anaturally-occurring nutritive protein. In some embodiments the secondpolypeptide sequence is selected from a tag for affinity purification, aprotein domain linker, and a protease recognition site. In someembodiments the tag for affinity purification is a polyhistidine-tag. Insome embodiments the protein domain linker comprises at least one copyof the sequence GGSG. In some embodiments the protease is selected frompepsin, trypsin, and chymotrypsin.

In some embodiments the recombinant nutritive protein consists of thefirst polypeptide sequence and the second polypeptide sequence.

In some embodiments the nutritive protein further comprises a thirdpolypeptide sequence comprising a fragment of at least 25 or at least 50amino acids of a naturally-occurring nutritive protein. In someembodiments the first and third polypeptide sequences are the same. Insome embodiments the first and third polypeptide sequences aredifferent. In some embodiments the first and third polypeptide sequencesare derived from the same naturally-occurring nutritive protein. In someembodiments the order of the first and third polypeptide sequences inthe isolated recombinant nutritive protein is the same as the order ofthe first and third polypeptide sequences in the naturally-occurringnutritive protein. In some embodiments the order of the first and thirdpolypeptide sequences in the nutritive protein is different than theorder of the first and third polypeptide sequences in thenaturally-occurring nutritive protein. In some embodiments the first andthird polypeptide sequences are derived from differentnaturally-occurring nutritive proteins. In some embodiments the secondpolypeptide sequence is flanked by the first and third polypeptidesequences.

In some embodiments of the nutritive protein, the first and/or thirdpolypeptide sequence comprises a fragment of a naturally-occurringnutritive protein and the ratio of branch chain amino acid residues tototal amino acid residues present in the first and/or third polypeptidesequence is equal to or greater than the ratio of branch chain aminoacid residues to total amino acid residues present in at least one ofwhey protein, egg protein, and soy protein.

In some embodiments of the nutritive protein, the first and/or thirdpolypeptide sequence comprises a first and/or third polypeptide sequencecomprising a fragment of a naturally-occurring nutritive protein and theratio of L residues to total amino acid residues present in the firstand/or third polypeptide sequence is equal to or greater than the ratioof L residues to total amino acid residues present in at least one ofwhey protein, egg protein, and soy protein.

In some embodiments of the nutritive protein, the first and/or thirdpolypeptide sequence comprises a first and/or third polypeptide sequencecomprising a fragment of a naturally-occurring nutritive protein and theratio of essential amino acid residues to total amino acid residuespresent in the first and/or third polypeptide sequence is equal to orgreater than the ratio of essential amino acid residues to total aminoacid residues present in at least one of whey protein, egg protein, andsoy protein.

In some embodiments of the nutritive protein, the first and/or thirdpolypeptide sequence comprises a first and/or third polypeptide sequencecomprising a fragment of a naturally-occurring nutritive protein and theratio of branch chain amino acid residues to total amino acid residuespresent in the first and/or third polypeptide sequence is equal to orgreater than the ratio of branch chain amino acid residues to totalamino acid residues present in at least one of whey protein, eggprotein, and soy protein; and the first and/or third polypeptidesequence comprises a ratio of L residues to total amino acid residuesthat is equal to or greater than the ratio of L residues to total aminoacid residues present in at least one of whey protein, egg protein, andsoy protein. In some such embodiments the first and/or third polypeptidesequence further comprises a ratio of essential amino acid residues tototal amino acid residues that is equal to or greater than the ratio ofessential amino acid residues to total amino acid residues present in atleast one of whey protein, egg protein, and soy protein.

In some embodiments of the nutritive protein, the first and/or thirdpolypeptide sequence comprises a first and/or third polypeptide sequencecomprising a ratio of branch chain amino acid residues to total aminoacid residues equal to or greater than 24% and a ratio of L residues tototal amino acid residues that is equal to or greater than 11%. In somesuch embodiments the first and/or third polypeptide sequence furthercomprises a ratio of essential amino acid residues to total amino acidresidues equal to or greater than 49%.

In some embodiments the naturally-occurring nutritive protein from whicha fragment is derived is a nutritive protein other than at least onenutritive protein selected from egg proteins such as ovalbumin,ovotransferrin, and ovomucuoid; meat proteins such as myosin, actin,tropomyosin, collagen, and troponin; milk proteins such as whey andcasein; cereal proteins such as casein, alpha1 casein, alpha2 casein,beta casein, kappa casein, beta-lactoglobulin, alpha-lactalbumin,glycinin, beta-conglycinin, glutelin, prolamine, gliadin, glutenin,albumin, globulin; chicken muscle proteins such as albumin, enolase,creatine kinase, phosphoglycerate mutase, triosephosphate isomerase,apolipoprotein, ovotransferrin, phosphoglucomutase, phosphoglyceratekinase, glycerol-3-phosphate dehydrogenase, glyceraldehyde 3-phosphatedehydrogenase, hemoglobin, cofilin, glycogen phosphorylase,fructose-1,6-bisphosphatase, actin, myosin, tropomyosin a-chain, caseinkinase, glycogen phosphorylase, fructose-1,6-bisphosphatase, aldolase,tubulin, vimentin, endoplasmin, lactate dehydrogenase, destrin,transthyretin, fructose bisphosphate aldolase, carbonic anhydrase,aldehyde dehydrogenase, annexin, adenosyl homocysteinase; pork muscleproteins such as actin, myosin, enolase, titin, cofilin,phosphoglycerate kinase, enolase, pyruvate dehydrogenase, glycogenphosphorylase, triosephosphate isomerase, myokinase; and fish proteinssuch as parvalbumin, pyruvate dehydrogenase, desmin, and triosephosphateisomerase.

Phenylketonuria (PKU) is an autosomal recessive metabolic geneticdisorder characterized by a mutation in the gene for the hepatic enzymephenylalanine hydroxylase (PAH), rendering it nonfunctional. This enzymeis necessary to metabolize phenylalanine to tyrosine. When PAH activityis reduced, phenylalanine accumulates and is converted intophenylpyruvate (also known as phenylketone), which is detected in theurine. Untreated children are normal at birth, but fail to attain earlydevelopmental milestones, develop microcephaly, and demonstrateprogressive impairment of cerebral function. Hyperactivity, EEGabnormalities and seizures, and severe learning disabilities are majorclinical problems later in life. A characteristic odor of skin, hair,sweat and urine (due to phenylacetate accumulation); and a tendency tohypopigmentation and eczema are also observed. All PKU patients mustadhere to a special diet low in Phe. Accordingly, nutritive proteinscomprising a low number or no Phe residues are desirable for PKUpatients. Such proteins can be obtained by selecting nutritive proteinsprovided herein that have few or no Phe residues. Accordingly, in someembodiments the nutritive protein comprises a ratio of Phe residues tototal amino acid residues equal to or lower than 5%, 4%, 3%, 2%, or 1%.In some embodiments the nutritive protein comprises 10 or fewer Pheresidues, 9 or fewer Phe residues, 8 or fewer Phe residues, 7 or fewerPhe residues, 6 or fewer Phe residues, 5 or fewer Phe residues, 4 orfewer Phe residues, 3 or fewer Phe residues, 2 or fewer Phe residues, 1Phe residue, or no Phe residues. In some embodiments, the nutritiveprotein comprises no Phe residues.

Arginine is a conditionally nonessential amino acid, meaning most of thetime it can be manufactured by the human body, and does not need to beobtained directly through the diet. Individuals who have poor nutrition,the elderly, or people with certain physical conditions (e.g., sepsis)may not produce sufficient amounts of arginine and therefore need toincrease their intake of foods containing arginine. Arginine is believedto have beneficial health properties, including reducing healing time ofinjuries (particularly bone), and decreasing blood pressure,particularly high blood pressure during high risk pregnancies(pre-eclampsia). In addition, studies have shown that dietarysupplementation with L-arginine is beneficial for enhancing thereproductive performance of pigs with naturally occurring in-trauterinegrowth retardation, enhancing protein deposition and postnatal growth ofmilk-fed piglets, normalizing plasma glucose levels instreptozotocin-induced diabetic rats, reducing fat mass in obese Zuckerdiabetic fatty (ZDF) rats, and improving vascular function in diabeticrats. In order to combine these benefits with at least one utility ofthe nutritive proteins disclosed herein, in some embodiments of thenutritive proteins disclosed herein the nutritive protein comprises aration of Arginine residues to total amino acid residues in thenutritive protein of equal to or greater than 3%, equal to or greaterthan 4%, equal to or greater than 5%, equal to or greater than 6%, equalto or greater than 7%, equal to or greater than 8%, equal to or greaterthan 9%, equal to or greater than 10%, equal to or greater than 11%, orequal to or greater than 12%.

Digestibility is a parameter relevant to the nutritive benefits andutility of nutritive proteins. Information relating to the relativecompleteness of digestion can serve as a predictor of peptidebioavailability (Daniel, H., 2003. Molecular and Integrative Physiologyof Intestinal Peptide Transport. Annual Review of Physiology, Volume 66,pp. 361-384). In some embodiments nutritive proteins disclosed hereinare screened to assess their digestibility. Digestibility of nutritiveproteins can be assessed by any suitable method known in the art. Insome embodiments digestibility is assessed by a physiologically relevantin vitro digestion reaction that includes one or both phases of proteindigestion, simulated gastric digestion and simulated intestinaldigestion (see, e.g., Moreno, et al., 2005. Stability of the majorallergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant invitro gastrointestinal digestion. FEBS Journal, pp. 341-352; Martos, G.,Contreras, P., Molina, E. & Lopez-Fandino, R., 2010. Egg White OvalbuminDigestion Mimicking Physiological Conditions. Journal of Agriculturaland food chemistry, pp. 5640-5648; Moreno, F. J., Mackie, A. R. & ClareMills, E. N., 2005. Phospholipid interactions protect the milk allergena-Lactalbumin from proteolysis during in vitro digestion. Journal ofagricultural and food chemistry, pp. 9810-9816). Briefly, test proteinsare sequentially exposed to a simulated gastric fluid (SGF) for 120minutes (the length of time it takes 90% of a liquid meal to pass fromthe stomach to the small intestine; see Kong, F. & Singh, R. P., 2008.Disintegration of Solid Foods in Human Stomach. Journal of Food Science,pp. 67-80) and then transferred to a simulated duodenal fluid (SDF) todigest for an additional 120 minutes. Samples at different stages of thedigestion (e.g., 2, 5, 15, 30, 60 and 120 min) are analyzed byelectrophoresis (e.g., chip electrophoresis or SDS-PAGE) to monitor thesize and amount of intact protein as well as any large digestionfragments (e.g., larger than 4 kDa). The disappearance of protein overtime indicates the rate at which the protein is digested in the assay.By monitoring the amount of intact protein observed over time, thehalf-life (τ½) of digestion is calculated for SGF and, if intact proteinis detected after treatment with SGF, the τ½ of digestion is calculatedfor SIF. This assay can be used to assess comparative digestibility(i.e., against a benchmark protein such as whey) or to assess absolutedigestibility. In some embodiments the digestibility of the nutritiveprotein is higher (i.e., the SGF τ½ and/or SIF τ½ is shorter) than wheyprotein. In some embodiments the nutritive protein has a SGF τ½ of 30minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes orless, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutesor less or 1 minute or less. In some embodiments the nutritive proteinhas a SIF τ½ of 30 minutes or less, 20 minutes or less, 15 minutes orless, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3minutes or less, 2 minutes or less or 1 minute or less. In someembodiments the nutritive protein is not detectable in one or both ofthe SGF and SIF assays by 2 minutes, 5 minutes, 15 minutes, 30 minutes,60 minutes, or 120 minutes. In some embodiments the nutritive protein isdigested at a constant rate and/or at a controlled rate in one or bothof SGF and SIF. In such embodiments the rate of digestion of thenutritive protein may not be optimized for the highest possible rate ofdigestion. In such embodiments the rate of absorption of the proteinfollowing ingestion by a mammal may be slower and the total time periodover which absorption occurs following ingestion may be longer than fornutritive proteins of similar amino acid composition that are digestedat a faster initial rate in one or both of SGF and SIF. In someembodiments the nutritive protein is completely or substantiallycompletely digested in SGF. In some embodiments the nutritive protein issubstantially not digested or not digested by SGF; in most suchembodiments the protein is digested in SIF.

Assessing protein digestibility can also provide insight into aprotein's potential allergenicity, as proteins or large fragments ofproteins that are resistant to digestive proteases can have a higherrisk of causing an allergenic reaction (Goodman, R. E. et al., 2008.Allergenicity assessment of genetically modified crops—what makes sense?Nature Biotechnology, pp. 73-81). To detect and identify peptides toosmall for chip electrophoresis analysis, liquid chromatography and massspectrometry can be used. In SGF samples, peptides can be directlydetected and identified by LC/MS. SIF protein digestions may requirepurification to remove bile acids before detection and identification byLC/MS.

In some embodiments digestibility of a nutritive protein is assessed byidentification and quantification of digestive protease recognitionsites in the protein amino acid sequence. In some embodiments thenutritive protein comprises at least one protease recognition siteselected from a pepsin recognition site, a trypsin recognition site, anda chymotrypsin recognition site.

As used herein, a “pepsin recognition site” is any site in a polypeptidesequence that is experimentally shown to be cleaved by pepsin. In someembodiments it is a peptide bond after (i.e., downstream of) an aminoacid residue selected from Phe, Trp, Tyr, Leu, Ala, Glu, and Gln,provided that the following residue is not an amino acid residueselected from Ala, Gly, and Val.

As used herein, a “trypsin recognition site” is any site in apolypeptide sequence that is experimentally shown to be cleaved bytrypsin. In some embodiments it is a peptide bond after an amino acidresidue selected from Lys or Arg, provided that the following residue isnot a proline.

As used herein, a “chymotrypsin recognition site” is any site in apolypeptide sequence that is experimentally shown to be cleaved bychymotrypsin. In some embodiments it is a peptide bond after an aminoacid residue selected from Phe, Trp, Tyr, and Leu.

Disulfide bonded cysteine residues in a protein tend to reduce the rateof digestion of the protein compared to what it would be in the absenceof the disulfide bond. For example, it has been shown that the rate ofdigestion of the protein b-lactoglobulin is increased when its disulfidebridges are cleaved (I. M. Reddy, N. K. D. Kella, and J. E. Kinsella.“Structural and Conformational Basis of the Resistance ofB-Lactoglobulin to Peptic and Chymotryptic Digestion”. J. Agric. FoodChem. 1988, 36, 737-741). Accordingly, digestibility of a nutritiveprotein with fewer disulfide bonds tends to be higher than for acomparable nutritive protein with a greater number of disulfide bonds.In some embodiments the nutritive proteins disclosed herein are screenedto identify the number of cysteine residues present in each and inparticular to allow selection of a nutritive protein comprising arelatively low number of cysteine residues. For example, naturallyoccurring nutritive proteins or fragments may be identified thatcomprise a no Cys residues or that comprise a relatively low number ofCys residues, such as 10 or fewer Cys residues, 9 or fewer Cys residues,8 or fewer Cys residues, 7 or fewer Cys residues, 6 or fewer Cysresidues, 5 or fewer Cys residues, 4 or fewer Cys residues, 3 or fewerCys residues, 2 or fewer Cys residues, 1 Cys residue, or no Cysresidues. In some embodiments one or more Cys residues in a naturallyoccurring nutritive protein or fragment thereof is removed by deletionand/or by substitution with another amino acid. In some embodiments 1Cys residue is deleted or replaced, 1 or more Cys residues are deletedor replaced, 2 or more Cys residues are deleted or replaced, 3 or moreCys residues are deleted or replaced, 4 or more Cys residues are deletedor replaced, 5 or more Cys residues are deleted or replaced, 6 or moreCys residues are deleted or replaced, 7 or more Cys residues are deletedor replaced, 8 or more Cys residues are deleted or replaced, 9 or moreCys residues are deleted or replaced, or 10 or more Cys residues aredeleted or replaced. In some embodiments the nutritive protein of thisdisclosure comprises a ratio of Cys residues to total amino acidresidues equal to or lower than 5%, 4%, 3%, 2%, or 1%. In someembodiments the nutritive protein comprises 10 or fewer Cys residues, 9or fewer Cys residues, 8 or fewer Cys residues, 7 or fewer Cys residues,6 or fewer Cys residues, 5 or fewer Cys residues, 4 or fewer Cysresidues, 3 or fewer Cys residues, 2 or fewer Cys residues, 1 Cysresidue, or no Cys residues. In some embodiments, the nutritive proteincomprises 1 or fewer Cys residues. In some embodiments, the nutritiveprotein comprises no Cys residues.

Alternatively or in addition, disulfide bonds that are or may be presentin a nutritive protein may be removed. Disulfides can be removed usingchemical methods by reducing the disulfide to two thiol groups withreducing agents such as beta-mercaptoethanol, dithiothreitol (DTT), ortris(2-carboxyethyl)phosphine (TCEP). The thiols can then be covalentlymodified or “capped” with reagents such as iodoacetamide,N-ethylmaleimide, or sodium sulfite (see, e.g., Crankshaw, M. W. andGrant, G. A. 2001. Modification of Cysteine. Current Protocols inProtein Science. 15.1.1-15.1.18).

Eukaryotic proteins are often glycosylated, and the carbohydrate chainsthat are attached to proteins serve various functions. N-linked andO-linked glycosylation are the two most common forms of glycosylationoccurring in proteins. N-linked glycosylation is the attachment of asugar molecule to a nitrogen atom in an amino acid residue in a protein.N-linked glycosylation occurs at Asparagine and Arginine residues.O-linked glycosylation is the attachment of a sugar molecule to anoxygen atom in an amino acid residue in a protein. O-linkedglycosylation occurs at Threonine and Serine residues.

Glycosylated proteins are often more soluble than their un-glycosylatedforms. In terms of protein drugs, proper glycosylation usually confershigh activity, proper antigen binding, better stability in the blood,etc. However, glycosylation necessarily means that a protein “carrieswith it” sugar moieties. Such sugar moieties may reduce the usefulnessof the nutritive proteins of this disclosure including recombinantnutritive proteins. For example, as demonstrated in the examples, acomparison of digestion of glycosylated and non-glycosylated forms ofthe same proteins shows that the non-glycosylated forms are digestedmore quickly than the glycosylated forms. For these reasons, in someembodiments the nutritive proteins according to the disclosure compriselow or no glycosylation. For example, in some embodiments the nutritiveproteins comprise a ratio of non-glycosilated to total amino acidresidues of at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%. In someembodiments the nutritive proteins to not comprise any glycosylation.

In some embodiments, the nutritive protein according to the disclosureis de-glycosylated after it is produced or after it is isolated.Nutritive proteins of low or no glycosylation may be made by any methodknown in the art. For example, enzymatic and/or chemical methods may beused (Biochem. J. (2003) 376, p 339-350.). Enzymes are producedcommercially at research scales for the removal of N-linked and O-linkedoligosaccharides. Chemical methods include use oftrifluoromethanesulfonic acid to selectively break N-linked and O-linkedpeptide-saccharide bonds. This method often results in a more completedeglycosylation than does the use of enzymatic methods.

In other embodiments, the nutritive protein according to the disclosureis produced with low or no glycosylation by a host organism. Mostbacteria and other prokaryotes have very limited capabilities toglycosylate proteins, especially heterologous proteins. Accordingly, insome embodiments of this disclosure a nutritive protein is maderecombinantly in a microorganism such that the level of glycosylation ofthe recombinant protein is low or no glycosylation. In some embodimentsthe level of glycosylation of the recombinant nutritive protein is lowerthan the level of glycosylation of the protein as it occurs in theorganism from which it is derived.

In some embodiments a nutritive protein or polypeptide according to thedisclosure comprises a ratio of amino acids selected from Asn, Arg, Ser,and Thr to total amino acids of 20% or less, 19% or less, 18% or less,17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% orless, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6%or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less. Insome embodiments, a nutritive protein or polypeptide according to thedisclosure comprises no amino acids selected from Asn, Arg, Ser, andThr. In some embodiments a nutritive protein or polypeptide according tothe disclosure comprises fewer than 20, fewer than 19, fewer than 18,fewer than 17, fewer than 16, fewer than 15, fewer than 14, fewer than13, fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewerthan 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewerthan 3, fewer than 2, fewer than 1, or no amino acids selected from Asn,Arg, Ser, and Thr.

In some embodiments of the nutritive proteins disclosed herein thenutritive protein is soluble. Solubility can be measured by any methodknown in the art. In some embodiments solubility is examined bycentrifuge concentration followed by protein concentration assays.Samples of nutritive proteins in 20 mM HEPES pH 7.5 are tested forprotein concentration according to protocols using two methods,Coomassie Plus (Bradford) Protein Assay (Thermo Scientific) andBicinchoninic Acid (BCA) Protein Assay (Sigma-Aldrich). Based on thesemeasurements 10 mg of protein is added to an Amicon Ultra 3 kDacentrifugal filter (Millipore). Samples are concentrated bycentrifugation at 10,000×g for 30 minutes. The final, now concentrated,samples are examined for precipitated protein and then tested forprotein concentration as above using two methods, Bradford and BCA.

In some embodiments the nutritive proteins have a final solubility limitof at least 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, or 100 g/L atphysiological pH. In some embodiments the nutritive proteins are greaterthan 50%, greater than 60%, greater than 70%, greater than 80%, greaterthan 90%, greater than 95%, greater than 96%, greater than 97%, greaterthan 98%, greater than 99%, or greater than 99.5% soluble with noprecipitated protein observed at a concentration of greater than 5 g/L,or 10 g/L, or 20 g/L, or 30 g/L, or 40 g/L, or 50 g/L, or 100 g/L atphysiological pH. In some embodiments, the solubility of the nutritiveprotein is higher than those typically reported in studies examining thesolubility limits of whey (12.5 g/L; Pelegrine et al., Lebensm.-Wiss.U.-Technol. 38 (2005) 77-80) and soy (10 g/L; Lee et al., JAOCS 80(1)(2003) 85-90).

As used herein, a “stable” protein is one that resists changes (e.g.,unfolding, oxidation, aggregation, hydrolysis, etc.) that alter thebiophysical (e.g., solubility), biological (e.g., digestibility), orcompositional (e.g. proportion of Leucine amino acids) traits of theprotein of interest.

Protein stability can be measured using various assays known in the artand nutritive proteins disclosed herein and having stability above athreshold can be selected. In some embodiments a protein is selectedthat displays thermal stability that is comparable to or better thanthat of whey protein. Thermal stability is a property that can helppredict the shelf life of a nutritive protein. In some embodiments ofthe assay stability of nutritive protein samples is determined bymonitoring aggregation formation using size exclusion chromatography(SEC) after exposure to extreme temperatures. Aqueous samples of theprotein to be tested are placed in a heating block at 90° C. and samplesare taken after 0, 1, 5, 10, 30 and 60 min for SEC analysis. Protein isdetected by monitoring absorbance at 214 nm, and aggregates arecharacterized as peaks eluting faster than the protein of interest. Nooverall change in peak area indicates no precipitation of protein duringthe heat treatment. Whey protein has been shown to rapidly form ˜80%aggregates when exposed to 90° C. in such an assay.

In some embodiments the thermal stability of a nutritive protein isdetermined by heating a sample slowly from 25° C. to 95° C. in presenceof a hydrophobic dye (e.g., ProteoStat® Thermal shift stability assaykit, Enzo Life Sciences) that binds to aggregated proteins that areformed as the protein denatures with increasing temperature (Niesen, F.H., Berglund, H. & Vadadi, M., 2007. The use of differential scanningfluorimetry to detect ligand interactions that promote proteinstability. Nature Protocols, Volume 2, pp. 2212-2221). Upon binding, thedye's fluorescence increases significantly, which is recorded by anrtPCR instrument and represented as the protein's melting curve(Lavinder, J. J., Hari, S. B., Suillivan, B. J. & Magilery, T. J., 2009.High-Throughput Thermal Scanning: A General, Rapid Dye-Binding ThermalShift Screen for Protein Engineering. Journal of the American ChemicalSociety, pp. 3794-3795). After the thermal shift is complete, samplesare examined for insoluble precipitates and further analyzed byanalytical size exclusion chromatography (SEC).

In some embodiments a nutritive protein of this disclosure showsresistance to aggregation, exhibiting, for example, less than 80%aggregation, 10% aggregation, or no detectable aggregation at elevatedtemperatures (e.g., 50° C., 60° C., 70° C., 80° C., 85° C., 90° C., or95° C.).

One benefit of stable nutritive proteins as disclosed herein is thatthey may be able to be stored for an extended period of time before use,in some instances without the need for refrigeration or cooling. In someembodiments, nutritive proteins are processed into a dry form (e.g., bylyophilization). In some embodiments, nutritive proteins are stable uponlyophilization. In some embodiments, such lyophilized nutritive proteinsmaintain their stability upon reconstitution (e.g., liquid formulation).

For most embodiments it is preferred that the nutritive protein notexhibit inappropriately high allergenicity. Accordingly, in someembodiments the potential allergenicy of the nutritive protein isassessed. This can be done by any suitable method known in the art. Insome embodiments an allergenicity score is calculated. The allergenicityscore is a primary sequence based metric based on WHO recommendations(http://www.fao.org/ag/agn/food/pdf/allergygm.pdf) for assessing howsimilar a protein is to any known allergen, the primary hypothesis beingthat high percent identity between a target and a known allergen islikely indicative of cross reactivity. For a given protein, thelikelihood of eliciting an allergic response can be assessed via one orboth of a complimentary pair of sequence homology based tests. The firsttest determines the protein's percent identity across the entiresequence via a global-global sequence alignment to a database of knownallergens using the FASTA algorithm with the BLOSUM50 substitutionmatrix, a gap open penalty of 10, and a gap extension penalty of 2. Ithas been suggested that proteins with less than 50% global homology areunlikely to be allergenic (Goodman R. E. et al. Allergenicity assessmentof genetically modified crops-what makes sense? Nat. Biotech. 26, 73-81(2008); Aalberse R. C. Structural biology of allergens. J. Allergy Clin.Immunol. 106, 228-238 (2000)).

In some embodiments of a nutritive protein, the nutritive protein hasless than 50% global homology to any known allergen in the database usedfor the analysis. In some embodiments a cutoff of less than 40% homologyis used. In some embodiments a cutoff of less than 30% homology is used.In some embodiments a cutoff of less than 20% homology is used. In someembodiments a cutoff of less than 10% homology is used. In someembodiments a cutoff of from 40% to 50% is used. In some embodiments acutoff of from 30% to 50% is used. In some embodiments a cutoff of from20% to 50% is used. In some embodiments a cutoff of from 10% to 50% isused. In some embodiments a cutoff of from 5% to 50% is used. In someembodiments a cutoff of from 0% to 50% is used. In some embodiments acutoff of greater than 50% global homology to any known allergen in thedatabase used for the analysis is used. In some embodiments a cutoff offrom 50% to 60% is used. In some embodiments a cutoff of from 50% to 70%is used. In some embodiments a cutoff of from 50% to 80% is used. Insome embodiments a cutoff of from 50% to 90% is used. In someembodiments a cutoff of from 55% to 60% is used. In some embodiments acutoff of from 65% to 70% is used. In some embodiments a cutoff of from70% to 75% is used. In some embodiments a cutoff of from 75% to 80% isused.

The second test assesses the local allergenicity along the proteinsequence by determining the local allergenicity of all possiblecontiguous 80 amino acid fragments via a global-local sequence alignmentof each fragment to a database of known allergens using the FASTAalgorithm with the BLOSUM50 substitution matrix, a gap open penalty of10, and a gap extension penalty of 2. The highest percent identity ofany 80 amino acid window with any allergen is taken as the final scorefor the protein of interest. The WHO guidelines suggest using a 35%identity cutoff with this fragment test. In some embodiments of anutritive protein, all possible fragments of the nutritive protein haveless than 35% local homology to any known allergen in the database usedfor the analysis using this test. In some embodiments a cutoff of lessthan 30% homology is used. In some embodiments a cutoff of from 30% to35% homology is used. In some embodiments a cutoff of from 25% to 30%homology is used. In some embodiments a cutoff of from 20% to 25%homology is used. In some embodiments a cutoff of from 15% to 20%homology is used. In some embodiments a cutoff of from 10% to 15%homology is used. In some embodiments a cutoff of from 5% to 10%homology is used. In some embodiments a cutoff of from 0% to 5% homologyis used. In some embodiments a cutoff of greater than 35% homology isused. In some embodiments a cutoff of from 35% to 40% homology is used.In some embodiments a cutoff of from 40% to 45% homology is used. Insome embodiments a cutoff of from 45% to 50% homology is used. In someembodiments a cutoff of from 50% to 55% homology is used. In someembodiments a cutoff of from 55% to 60% homology is used. In someembodiments a cutoff of from 65% to 70% homology is used. In someembodiments a cutoff of from 70% to 75% homology is used. In someembodiments a cutoff of from 75% to 80% homology is used.

Skilled artisans are able to identify and use a suitable database ofknown allergens for this purpose. In some embodiments the database iscustom made by selecting proteins from more than one database source. Insome embodiments the custom database comprises pooled allergen listscollected by the Food Allergy Research and Resource Program(http://www.allergenonline.org/), UNIPROT annotations(http://www.uniprot.org/docs/allergen), and the Structural Database ofAllergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/sdap_lnk.html).This database includes all currently recognized allergens by theInternational Union of Immunological Societies (IUIS,http://www.allergen.org/) as well as a large number of additionalallergens not yet officially named. In some embodiments the databasecomprises a subset of known allergen proteins available in knowndatabases; that is, the database is a custom selected subset of knownallergen proteins. In some embodiments the database of known allergenscomprises at least 10 proteins, at least 20 proteins, at least 30proteins, at least 40 proteins, at least 50 proteins, at least 100,proteins, at least 200 proteins, at least 300 proteins, at least 400proteins, at least 500 proteins, at least 600 proteins, at least 700proteins, at least 800 proteins, at least 900 proteins, at least 1,000proteins, at least 1,100 proteins, at least 1,200 proteins, at least1,300 proteins, at least 1,400 proteins, at least 1,500 proteins, atleast 1,600 proteins, at least 1,700 proteins, at least 1,800 proteins,at least 1,900 proteins, or at least 2,000 proteins. In some embodimentsthe database of known allergens comprises from 100 to 500 proteins, from200 to 1,000 proteins, from 500 to 1,000 proteins, from 500 to 1,000proteins, or from 1,000 to 2,000 proteins.

In some embodiments all (or a selected subset) of contiguous amino acidwindows of different lengths (e.g., 70, 60, 50, 40, 30, 20, 10, 8 or 6amino acid windows) of a nutritive protein are tested against theallergen database and peptide sequences that have 100% identity, 95% orhigher identity, 90% or higher identity, 85% or higher identity, 80% orhigher identity, 75% or higher identity, 70% or higher identity, 65% orhigher identity, 60% or higher identity, 55% or higher identity, or 50%or higher identity matches are identified for further examination ofpotential allergenicity.

Another method of predicting the allergenicity of a protein is to assessthe homology of the protein to a protein of human origin. The humanimmune system is exposed to a multitude of possible allergenic proteinson a regular basis and has the intrinsic ability to differentiatebetween the host body's proteins and exogenous proteins. The exactnature of this ability is not always clear, and there are many diseasesthat arise as a result of the failure of the body to differentiate selffrom non-self (e.g. arthritis). Nonetheless, the fundamental hypothesisis that proteins that share a degree of sequence homology to humanproteins are less likely to elicit an immune response. In particular, ithas been shown that for some protein families with known allergenicmembers (tropomyosins, parvalbumins, caseins), those proteins that bearmore sequence homology to their human counterparts relative to knownallergenic proteins, are not thought to be allergenic (Jenkins J. A. etal. Evolutionary distance from human homologs reflects allergenicity ofanimal food proteins. J. Allergy Clin Immunol. 120 (2007): 1399-1405).For a given protein, a human homology score is measured by determiningthe maximum percent identity of the protein to a database of humanproteins (e.g., the UNIPROT database) from a global-local alignmentusing the FASTA algorithm with the BLOSUM50 substitution matrix, a gapopen penalty of 10, and a gap extension penalty of 2. According toJenkins et al. (Jenkins J. A. et al. Evolutionary distance from humanhomologs reflects allergenicity of animal food proteins. J. Allergy ClinImmunol. 120 (2007): 1399-1405) proteins with a sequence identity to ahuman protein above about 62% are less likely to be allergenic. Skilledartisans are able to identify and use a suitable database of known humanproteins for this purpose, for example, by searching the UNIPROTdatabase (http://www.uniprot.org). In some embodiments the database iscustom made by selecting proteins from more than one database source. Ofcourse the database may but need not be comprehensive. In someembodiments the database comprises a subset of human proteins; that is,the database is a custom selected subset of human proteins. In someembodiments the database of human proteins comprises at least 10proteins, at least 20 proteins, at least 30 proteins, at least 40proteins, at least 50 proteins, at least 100, proteins, at least 200proteins, at least 300 proteins, at least 400 proteins, at least 500proteins, at least 600 proteins, at least 700 proteins, at least 800proteins, at least 900 proteins, at least 1,000 proteins, at least 2,000proteins, at least 3,000 proteins, at least 4,000 proteins, at least5,000 proteins, at least 6,000 proteins, at least 7,000 proteins, atleast 8,000 proteins, at least 9,000 proteins, or at least 10,000proteins. In some embodiments the database comprises from 100 to 500proteins, from 200 to 1,000 proteins, from 500 to 1,000 proteins, from500 to 1,000 proteins, from 1,000 to 2,000 proteins, from 1,000 to 5,000proteins, or from 5,000 to 10,000 proteins. In some embodiments thedatabase comprises at least 90%, at least 95%, or at least 99% of allknown human proteins.

In some embodiments of a nutritive protein, the nutritive protein is atleast 20% homologous to a human protein. In some embodiments a cutoff ofat least 30% homology is used. In some embodiments a cutoff of at least40% homology is used. In some embodiments a cutoff of at least 50%homology is used. In some embodiments a cutoff of at least 60% homologyis used. In some embodiments a cutoff of at least 70% homology is used.In some embodiments a cutoff of at least 80% homology is used. In someembodiments a cutoff of at least 62% homology is used. In someembodiments a cutoff of from at least 20% homology to at least 30%homology is used. In some embodiments a cutoff of from at least 30%homology to at least 40% homology is used. In some embodiments a cutoffof from at least 50% homology to at least 60% homology is used. In someembodiments a cutoff of from at least 60% homology to at least 70%homology is used. In some embodiments a cutoff of from at least 70%homology to at least 80% homology is used.

For most embodiments it is preferred that the nutritive protein notexhibit inappropriately high toxicity. Accordingly, in some embodimentsthe potential toxicity of the nutritive protein is assessed. This can bedone by any suitable method known in the art. In some embodiments atoxicity score is calculated by determining the protein's percentidentity to databases of known toxic proteins (e.g., toxic proteinsidentified from the UNIPROT database). A global-global alignment of theprotein of interest against the database of known toxins is performedusing the FASTA algorithm with the BLOSUM50 substitution matrix, a gapopen penalty of 10, and a gap extension penalty of 2. In someembodiments of a nutritive protein, the nutritive protein is less than35% homologous to a known toxin. In some embodiments a cutoff of lessthan 35% homology is used. In some embodiments a cutoff of from 30% to35% homology is used. In some embodiments a cutoff of from 25% to 35%homology is used. In some embodiments a cutoff of from 20% to 35%homology is used. In some embodiments a cutoff of from 15% to 35%homology is used. In some embodiments a cutoff of from 10% to 35%homology is used. In some embodiments a cutoff of from 5% to 35%homology is used. In some embodiments a cutoff of from 0% to 35%homology is used. In some embodiments a cutoff of greater than 35%homology is used. In some embodiments a cutoff of from 35% to 40%homology is used. In some embodiments a cutoff of from 35% to 45%homology is used. In some embodiments a cutoff of from 35% to 50%homology is used. In some embodiments a cutoff of from 35% to 55%homology is used. In some embodiments a cutoff of from 35% to 60%homology is used. In some embodiments a cutoff of from 35% to 70%homology is used. In some embodiments a cutoff of from 35% to 75%homology is used. In some embodiments a cutoff of from 35% to 80%homology is used. Skilled artisans are able to identify and use asuitable database of known toxins for this purpose, for example, bysearching the UNIPROT database (http://www.uniprot.org). In someembodiments the database is custom made by selecting proteins identifiedas toxins from more than one database source. In some embodiments thedatabase comprises a subset of known toxic proteins; that is, thedatabase is a custom selected subset of known toxic proteins. In someembodiments the database of toxic proteins comprises at least 10proteins, at least 20 proteins, at least 30 proteins, at least 40proteins, at least 50 proteins, at least 100, proteins, at least 200proteins, at least 300 proteins, at least 400 proteins, at least 500proteins, at least 600 proteins, at least 700 proteins, at least 800proteins, at least 900 proteins, at least 1,000 proteins, at least 2,000proteins, at least 3,000 proteins, at least 4,000 proteins, at least5,000 proteins, at least 6,000 proteins, at least 7,000 proteins, atleast 8,000 proteins, at least 9,000 proteins, or at least 10,000proteins. In some embodiments the database comprises from 100 to 500proteins, from 200 to 1,000 proteins, from 500 to 1,000 proteins, from500 to 1,000 proteins, from 1,000 to 2,000 proteins, from 1,000 to 5,000proteins, or from 5,000 to 10,000 proteins.

For some embodiments it is preferred that the nutritive protein notexhibit anti-nutritional activity (“anti-nutricity”), i.e., proteinsthat have the potential to prevent the absorption of nutrients fromfood. Examples of anti-nutritive factors include protease inhibitors,which inhibit the actions of trypsin, pepsin and other proteases in thegut, preventing the digestion and subsequent absorption of protein.Accordingly, in some embodiments the potential anti-nutricity of thenutritive protein is assessed. This can be done by any suitable methodknown in the art. In some embodiments an anti-nutricity score iscalculated by determining the protein's percent identity to databases ofknown protease inhibitors (e.g., protease inhibitors identified from theUNIPROT database). A global-global alignment of the protein of interestagainst the database of known protease inhibitors is performed using theFASTA algorithm with the BLOSUM50 substitution matrix, a gap openpenalty of 10, and a gap extension penalty of 2, to identify whether thenutritive protein is homologous to a known anti-nutritive protein. Insome embodiments of a nutritive protein, the nutritive protein has lessthan 35% global homology to any known anti-nutritive protein (e.g., anyknown protease inhibitor) in the database used for the analysis. In someembodiments a cutoff of less than 35% identify is used. In someembodiments a cutoff of from 30% to 35% is used. In some embodiments acutoff of from 25% to 35% is used. In some embodiments a cutoff of from20% to 35% is used. In some embodiments a cutoff of from 15% to 35% isused. In some embodiments a cutoff of from 10% to 35% is used. In someembodiments a cutoff of from 5% to 35% is used. In some embodiments acutoff of from 0% to 35% is used. In some embodiments a cutoff ofgreater than 35% identify is used. In some embodiments a cutoff of from35% to 40% is used. In some embodiments a cutoff of from 35% to 45% isused. In some embodiments a cutoff of from 35% to 50% is used. In someembodiments a cutoff of from 35% to 55% is used. In some embodiments acutoff of from 35% to 60% is used. In some embodiments a cutoff of from35% to 70% is used. In some embodiments a cutoff of from 35% to 75% isused. In some embodiments a cutoff of from 35% to 80% is used. Skilledartisans are able to identify and use a suitable database of knownprotease inhibitors for this purpose, for example, by searching theUNIPROT database (http://www.uniprot.org). In some embodiments thedatabase is custom made by selecting proteins identifiedprotease-inhibitors as from more than one database source. In someembodiments the database comprises a subset of known protease inhibitorsavailable in databases; that is, the database is a custom selectedsubset of known protease inhibitor proteins. In some embodiments thedatabase of known protease inhibitor proteins comprises at least 10proteins, at least 20 proteins, at least 30 proteins, at least 40proteins, at least 50 proteins, at least 100, proteins, at least 200proteins, at least 300 proteins, at least 400 proteins, at least 500proteins, at least 600 proteins, at least 700 proteins, at least 800proteins, at least 900 proteins, at least 1,000 proteins, at least 1,100proteins, at least 1,200 proteins, at least 1,300 proteins, at least1,400 proteins, at least 1,500 proteins, at least 1,600 proteins, atleast 1,700 proteins, at least 1,800 proteins, at least 1,900 proteins,or at least 2,000 proteins. In some embodiments the database of knownprotease inhibitor proteins comprises from 100 to 500 proteins, from 200to 1,000 proteins, from 500 to 1,000 proteins, from 500 to 1,000proteins, or from 1,000 to 2,000 proteins, or from 2,000 to 3,000proteins.

In other embodiments a nutritive protein that does exhibit some degreeof protease inhibitor activity is used. For example, in some embodimentssuch a protein may be useful because it delays protease digestion whenthe nutritive protein is consumed such that the nutritive proteintraverse a greater distance within the GI tract before it is digested,thus delaying absorption. For example, in some embodiments the nutritiveprotein inhibits gastric digestion but not intestinal digestion.

Delaney B. et al. (Evaluation of protein safety in the context ofagricultural biotechnology. Food. Chem. Toxicol. 46 (2008: S71-S97))suggests that one should avoid both known toxic and anti-nutritiveproteins when assessing the safety of a possible food protein. In someembodiments of a nutritive protein, the nutritive protein has afavorably low level of global homology to a database of known toxicproteins and/or a favorably low level of global homology to a databaseof known anti-nutricity proteins (e.g., protease inhibitors), as definedherein.

One feature that can enhance the utility of a nutritive protein is itscharge (or per amino acid charge). Nutritive proteins with higher chargecan in some embodiments exhibit desirable characteristics such asincreased solubility, increased stability, resistance to aggregation,and desirable taste profiles. For example, a charged nutritive proteinthat exhibits enhanced solubility can be formulated into a beverage orliquid formulation that includes a high concentration of nutritiveprotein in a relatively low volume of solution, thus delivering a largedose of protein nutrition per unit volume. A charged nutritive proteinthat exhibits enhanced solubility can be useful, for example, in sportsdrinks or recovery drinks wherein a user (e.g., an athlete) wants toingest nutritive protein before, during or after physical activity. Acharged nutritive protein that exhibits enhanced solubility can also beparticularly useful in a clinical setting wherein a subject (e.g., apatient or an elderly person) is in need of protein nutrition but isunable to ingest solid foods or large volumes of liquids.

For example, the net charge (ChargeP) of a polypeptide at pH 7 can becalculated using the following formula:

ChargeP=−0.002−(C)(0.045)−(D)(0.999)−(E)(0.998)+(H)(0.091)+(K)(1.0)+(R)(1.0)−(Y)(−0.001)

where C is the number of cysteine residues, D is the number of asparticacid residues, E is the number of glutamic acid residues, H is thenumber of histidine residues, K is the number of lysine residues, R isthe number of arginine residues and Y is the number of tyrosine residuesin the polypeptide. The per amino acid charge (ChargeA) of thepolypeptide can be calculated by dividing the net charge (ChargeP) bythe number of amino acid residues (N), i.e., ChargeA=ChargeP/N. (SeeBassi S (2007), “A Primer on Python for Life Science Researchers.” PLoSComput Biol 3(11): e199. doi:10.1371/journal.pcbi.0030199).

One metric for assessing the hydrophilicity and potential solubility ofa given protein is the solvation score. Solvation score is defined asthe total free energy of solvation (i.e. the free energy changeassociated with transfer from gas phase to a dilute solution) for allamino acid side chains if each residue were solvated independently,normalized by the total number of residues in the sequence. The sidechain solvation free energies are found computationally by calculatingthe electrostatic energy difference between a vacuum dielectric of 1 anda water dielectric of 80 (by solving the Poisson-Boltzmann equation) aswell as the non-polar, Van der Waals energy using a linear solventaccessible surface area model (D. Sitkoff, K. A. Sharp, B. Honig.“Accurate Calculation of Hydration Free Energies Using MacroscopicSolvent Models”. J. Phys. Chem. 98, 1994). For amino acids withionizable sidechains (Arg, Asp, Cys, Glu, His, Lys and Tyr), an averagesolvation free energy is used based on the relative probabilities foreach ionization state at the specified pH. Solvation scores start at 0and continue into negative values, and the more negative the solvationscore, the more hydrophilic and potentially soluble the protein ispredicted to be. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −10 or less at pH 7. In someembodiments of a nutritive protein, the nutritive protein has asolvation score of −15 or less at pH 7. In some embodiments of anutritive protein, the nutritive protein has a solvation score of −20 orless at pH 7. In some embodiments of a nutritive protein, the nutritiveprotein has a solvation score of −25 or less at pH 7. In someembodiments of a nutritive protein, the nutritive protein has asolvation score of −30 or less at pH 7. In some embodiments of anutritive protein, the nutritive protein has a solvation score of −35 orless at pH 7. In some embodiments of a nutritive protein, the nutritiveprotein has a solvation score of −40 or less at pH 7.

The solvation score is a function of pH by virtue of the pH dependenceof the molar ratio of undissociated weak acid ([HA]) to conjugate base([A−]) as defined by the Henderson-Hasselbalch equation:

${pH} = {{pKa} + {\log \mspace{11mu} \left( \frac{\left\lbrack A^{-} \right\rbrack}{\lbrack{HA}\rbrack} \right)}}$

All weak acids have different solvation free energies compared to theirconjugate bases, and the solvation free energy used for a given residuewhen calculating the solvation score at a given pH is the weightedaverage of those two values.

Accordingly, in some embodiments of a nutritive protein, the nutritiveprotein has a solvation score of −10 or less at an acidic pH. In someembodiments of a nutritive protein, the nutritive protein has asolvation score of −15 or less at an acidic pH. In some embodiments of anutritive protein, the nutritive protein has a solvation score of −20 orless at an acidic pH. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −25 or less at an acidic pH.In some embodiments of a nutritive protein, the nutritive protein has asolvation score of −30 or less at an acidic pH. In some embodiments of anutritive protein, the nutritive protein has a solvation score of −35 orless at an acidic pH. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −40 or less at acidic pH.

Accordingly, in some embodiments of a nutritive protein, the nutritiveprotein has a solvation score of −10 or less at a basic pH. In someembodiments of a nutritive protein, the nutritive protein has asolvation score of −15 or less at a basic pH. In some embodiments of anutritive protein, the nutritive protein has a solvation score of −20 orless at a basic pH. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −25 or less at a basic pH. Insome embodiments of a nutritive protein, the nutritive protein has asolvation score of −30 or less at a basic pH. In some embodiments of anutritive protein, the nutritive protein has a solvation score of −35 orless at a basic pH. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −40 or less at basic pH.

Accordingly, in some embodiments of a nutritive protein, the nutritiveprotein has a solvation score of −10 or less at a pH range selected from2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, and 11-12. In someembodiments of a nutritive protein, the nutritive protein has asolvation score of −15 or less at a pH range selected from 2-3, 3-4,4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, and 11-12. In some embodiments ofa nutritive protein, the nutritive protein has a solvation score of −20or less at a pH range selected from 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9,9-10, 10-11, and 11-12. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −25 or less at a pH rangeselected from 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, and 11-12.In some embodiments of a nutritive protein, the nutritive protein has asolvation score of −30 or less at a pH range selected from 2-3, 3-4,4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, and 11-12. In some embodiments ofa nutritive protein, the nutritive protein has a solvation score of −35or less at a pH range selected from 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9,9-10, 10-11, and 11-12. In some embodiments of a nutritive protein, thenutritive protein has a solvation score of −40 or less at a pH rangeselected from 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, and 11-12.

The aggregation score is a primary sequence based metric for assessingthe hydrophobicity and likelihood of aggregation of a given protein.Using the Kyte and Doolittle hydrophobity scale (Kyte J, Doolittle R F(May 1982) “A simple method for displaying the hydropathic character ofa protein”. J. Mol. Biol. 157 (1): 105-32), which gives hydrophobicresidues positive values and hydrophilic residues negative values, theaverage hydrophobicity of a protein sequence is calculated using amoving average of five residues. The aggregation score is drawn from theresulting plot by determining the area under the curve for valuesgreater than zero and normalizing by the total length of the protein.The underlying hypothesis is that aggregation is the result of two ormore hydrophobic patches coming together to exclude water and reducesurface exposure, and the likelihood that a protein will aggregate is afunction of how densely packed its hydrophobic (i.e., aggregation prone)residues are. Aggregation scores start at 0 and continue into positivevalues, and the smaller the aggregation score, the less hydrophobic andpotentially less prone to aggregation the protein is predicted to be. Insome embodiments of a nutritive protein, the nutritive protein has anaggregation score of 2 or less. In some embodiments of a nutritiveprotein, the nutritive protein has an aggregation score of 1.5 or less.In some embodiments of a nutritive protein, the nutritive protein has anaggregation score of 1 or less. In some embodiments of a nutritiveprotein, the nutritive protein has an aggregation score of 0.9 or less.In some embodiments of a nutritive protein, the nutritive protein has anaggregation score of 0.8 or less. In some embodiments of a nutritiveprotein, the nutritive protein has an aggregation score of 0.7 or less.In some embodiments of a nutritive protein, the nutritive protein has anaggregation score of 0.6 or less. In some embodiments of a nutritiveprotein, the nutritive protein has an aggregation score of 0.5 or less.In some embodiments of a nutritive protein, the nutritive protein has anaggregation score of 0.4 or less. In some embodiments of a nutritiveprotein, the nutritive protein has an aggregation score of 0.3 or less.In some embodiments of a nutritive protein, the nutritive protein has anaggregation score of 0.2 or less. In some embodiments of a nutritiveprotein, the nutritive protein has an aggregation score of 0.1 or less.

In some cases, soluble expression is desirable because it can increasethe amount and/or yield of the nutritive protein and facilitate one ormore of the isolation and purification of the nutritive protein. In someembodiments, the nutritive proteins of this disclosure are solublyexpressed in the host organism. Solvation score and aggregation scorecan be used to predict soluble expression of recombinant nutritiveproteins in a host organism. As shown in Example 8, this disclosureprovides evidence suggesting that nutritive proteins with solvationscores of ≦−20 and aggregation scores of ≦0.75 are more likely to berecombinantly expressed in a particular E. coli expression system.Moreover, the data also suggests that nutritive proteins with solvationscores of ≦−20 and aggregation scores of ≦0.5 are more likely to besolubly expressed in this system. Therefore, in some embodiments thenutritive protein of this disclosure has a solvation score of −20 orless. In some embodiments the nutritive protein has an aggregation scoreof 0.75 or less. In some embodiments the nutritive protein has anaggregation score of 0.5 or less. In some embodiments the nutritiveprotein has a solvation score of −20 or less and an aggregation score of0.75 or less. In some embodiments the nutritive protein has a solvationscore of −20 or less and an aggregation score of 0.5 or less.

Certain free amino acids and mixtures of free amino acids are known tohave a bitter or otherwise unpleasant taste. In addition, hydrolysatesof common proteins (e.g., whey and soy) often have a bitter orunpleasant taste. In some embodiments, nutritive proteins disclosed anddescribed herein do not have a bitter or otherwise unpleasant taste. Insome embodiments, nutritive proteins disclosed and described herein havea more acceptable taste as compared to at least one of free amino acids,mixtures of free amino acids, and/or protein hydrolysates. In someembodiments, nutritive proteins disclosed and described herein have ataste that is equal to or exceeds at least one of whey protein.

Proteins are known to have tastes covering the five established tastemodalities: sweet, sour, bitter, salty and umami. The taste of aparticular protein (or its lack thereof) can be attributed to severalfactors, including the primary structure, the presence of charged sidechains, and the electronic and conformational features of the protein.In some embodiments, nutritive proteins disclosed and described hereinare designed to have a desired taste (e.g., sweet, salty, umami) and/ornot to have an undesired taste (e.g., bitter, sour). In this context“design” includes, for example, selecting naturally occurring proteinsembodying features that achieve the desired taste property, as well ascreating muteins of naturally-occurring proteins that have desired tasteproperties. For example, nutritive proteins can be designed to interactwith specific taste receptors, such as sweet receptors (T1R2-T1R3heterodimer) or umami receptors (T1R1-T1R3 heterodimer, mGluR4, and/ormGluR1). Further, nutritive proteins may be designed not to interact, orto have diminished interaction, with other taste receptors, such asbitter receptors (T2R receptors).

Nutritive proteins disclosed and described herein can also elicitdifferent physical sensations in the mouth when ingested, sometimesreferred to as “mouth feel”. The mouth feel of the nutritive proteinsmay be due to one or more factors including primary structure, thepresence of charged side chains, and the electronic and conformationalfeatures of the protein. In some embodiments, nutritive proteins elicita buttery or fat-like mouth feel when ingested.

In some embodiments the nutritive protein comprises from 20 to 5,000amino acids, from 20-2,000 amino acids, from 20-1,000 amino acids, from20-500 amino acids, from 20-250 amino acids, from 20-200 amino acids,from 20-150 amino acids, from 20-100 amino acids, from 20-40 aminoacids, from 30-50 amino acids, from 40-60 amino acids, from 50-70 aminoacids, from 60-80 amino acids, from 70-90 amino acids, from 80-100 aminoacids, at least 25 amino acids, at least 30 amino acids, at least 35amino acids, at least 40 amino acids, at least 45 amino acids, at least50 amino acids, at least 55 amino acids, at least 60 amino acids, atleast 65 amino acids, at least 70 amino acids, at least 75 amino acids,at least 80 amino acids, at least 85 amino acids, at least 90 aminoacids, at least 95 amino acids, at least 100 amino acids, at least 105amino acids, at least 110 amino acids, at least 115 amino acids, atleast 120 amino acids, at least 125 amino acids, at least 130 aminoacids, at least 135 amino acids, at least 140 amino acids, at least 145amino acids, at least 150 amino acids, at least 155 amino acids, atleast 160 amino acids, at least 165 amino acids, at least 170 aminoacids, at least 175 amino acids, at least 180 amino acids, at least 185amino acids, at least 190 amino acids, at least 195 amino acids, atleast 200 amino acids, at least 205 amino acids, at least 210 aminoacids, at least 215 amino acids, at least 220 amino acids, at least 225amino acids, at least 230 amino acids, at least 235 amino acids, atleast 240 amino acids, at least 245 amino acids, or at least 250 aminoacids. In some embodiments the nutritive protein consists of from 20 to5,000 amino acids, from 20-2,000 amino acids, from 20-1,000 amino acids,from 20-500 amino acids, from 20-250 amino acids, from 20-200 aminoacids, from 20-150 amino acids, from 20-100 amino acids, from 20-40amino acids, from 30-50 amino acids, from 40-60 amino acids, from 50-70amino acids, from 60-80 amino acids, from 70-90 amino acids, from 80-100amino acids, at least 25 amino acids, at least 30 amino acids, at least35 amino acids, at least 40 amino acids, at least 2455 amino acids, atleast 50 amino acids, at least 55 amino acids, at least 60 amino acids,at least 65 amino acids, at least 70 amino acids, at least 75 aminoacids, at least 80 amino acids, at least 85 amino acids, at least 90amino acids, at least 95 amino acids, at least 100 amino acids, at least105 amino acids, at least 110 amino acids, at least 115 amino acids, atleast 120 amino acids, at least 125 amino acids, at least 130 aminoacids, at least 135 amino acids, at least 140 amino acids, at least 145amino acids, at least 150 amino acids, at least 155 amino acids, atleast 160 amino acids, at least 165 amino acids, at least 170 aminoacids, at least 175 amino acids, at least 180 amino acids, at least 185amino acids, at least 190 amino acids, at least 195 amino acids, atleast 200 amino acids, at least 205 amino acids, at least 210 aminoacids, at least 215 amino acids, at least 220 amino acids, at least 225amino acids, at least 230 amino acids, at least 235 amino acids, atleast 240 amino acids, at least 245 amino acids, or at least 250 aminoacids.

B. Nucleic Acids

Also provided herein are nucleic acids encoding nutritive polypeptidesor proteins. In some embodiments the nucleic acid is isolated. In someembodiments the nucleic acid is purified.

In some embodiments of the nucleic acid, the nucleic acid comprises anucleic acid sequence that encodes a first polypeptide sequencedisclosed in Section A above. In some embodiments of the nucleic acid,the nucleic acid consists of a nucleic acid sequence that encodes afirst polypeptide sequence disclosed in Section A above. In someembodiments of the nucleic acid, the nucleic acid comprises a nucleicacid sequence that encodes a nutritive protein disclosed in Section Aabove. In some embodiments of the nucleic acid, the nucleic acidconsists of a nucleic acid sequence that encodes a nutritive proteindisclosed in Section A above. In some embodiments of the nucleic acidthe nucleic acid sequence that encodes the first polypeptide sequence isoperatively linked to at least one expression control sequence. Forexample, in some embodiments of the nucleic acid the nucleic acidsequence that encodes the first polypeptide sequence is operativelylinked to a promoter such as a promoter described in Section D below.

Accordingly, in some embodiments the nucleic acid molecule of thisdisclosure encodes a polypeptide or protein that itself is a nutritivepolypeptide or protein. Such a nucleic acid molecule may be referred toas a “nutritive nucleic acid”. In some embodiments the nutritive nucleicacid encodes a polypeptide or protein that itself comprises at least oneof: a) a ratio of branch chain amino acid residues to total amino acidresidues of at least 24%; b) a ratio of Leu residues to total amino acidresidues of at least 11%; and c) a ratio of essential amino acidresidues to total amino acid residues of at least 49%. In someembodiments the nutritive nucleic acid comprises at least 10nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, atleast 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides,at least 100 nucleotides, at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, at least 900 nucleotides, at least 1,000 nucleotides. Insome embodiments the nutritive nucleic acid comprises from 10 to 100nucleotides, from 20 to 100 nucleotides, from 10 to 50 nucleotides, orfrom 20 to 40 nucleotides In some embodiments the nutritive nucleic acidcomprises all or part of an open reading frame that encodes a naturallyoccurring nutritive polypeptide or protein. In some embodiments thenutritive nucleic acid consists of an open reading frame that encodes afragment of a naturally occurring nutritive protein, wherein the openreading frame does not encode the complete naturally occurring nutritiveprotein.

In some embodiments the nutritive nucleic acid is a cDNA.

In some embodiments nucleic acid molecules are provided that comprise asequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 99.9% identical to a naturally occurring nutritive nucleicacid. In some embodiments nucleic acids are provided that hybridizeunder stringent hybridization conditions with at least one referencenutritive nucleic acid.

The nutritive nucleic acids and fragments thereof provided in thisdisclosure display utility in a variety of systems and methods. Forexample, the fragments may be used as probes in various hybridizationtechniques. Depending on the method, the target nucleic acid sequencesmay be either DNA or RNA. The target nucleic acid sequences may befractionated (e.g., by gel electrophoresis) prior to the hybridization,or the hybridization may be performed on samples in situ. One of skillin the art will appreciate that nucleic acid probes of known sequencefind utility in determining chromosomal structure (e.g., by Southernblotting) and in measuring gene expression (e.g., by Northern blotting).In such experiments, the sequence fragments are preferably detectablylabeled, so that their specific hydridization to target sequences can bedetected and optionally quantified. One of skill in the art willappreciate that the nucleic acid fragments of this disclosure may beused in a wide variety of blotting techniques not specifically describedherein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip:Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);Microarray Biochip: Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376).

C. Vectors

Also provided are vectors, including expression vectors, which compriseat least one of the nucleic acid molecules disclosed herein, asdescribed further herein. In some embodiments, the vectors comprise atleast one isolated nucleic acid molecule encoding a nutritive protein asdisclosed herein. In alternative embodiments, the vectors comprise sucha nucleic acid molecule operably linked to one or more expressioncontrol sequence. The vectors can thus be used to express at least onerecombinant protein in a recombinant microbial host cell.

Suitable vectors for expression of nucleic acids in microorganisms arewell known to those of skill in the art. Suitable vectors for use incyanobacteria are described, for example, in Heidorn et al., “SyntheticBiology in Cyanobacteria: Engineering and Analyzing Novel Functions,”Methods in Enzymology, Vol. 497, Ch. 24 (2011). Exemplary replicativevectors that can be used for engineering cyanobacteria as disclosedherein include pPMQAK1, pSL1211, pFC1, pSB2A, pSCR119/202, pSUN119/202,pRL2697, pRL25C, pRL1050, pSG111M, and pPBH201.

Other vectors such as pJB 161 which are capable of receiving nucleicacid sequences disclosed herein may also be used. Vectors such as pJB161 comprise sequences which are homologous with sequences present inplasmids endogenous to certain photosynthetic microorganisms (e.g.,plasmids pAQ1, pAQ3, and pAQ4 of certain Synechococcus species).Examples of such vectors and how to use them is known in the art andprovided, for example, in Xu et al., “Expression of Genes inCyanobacteria: Adaptation of Endogenous Plasmids as Platforms forHigh-Level Gene Expression in Synechococcus sp. PCC 7002,” Chapter 21 inRobert Carpentier (ed.), “Photosynthesis Research Protocols,” Methods inMolecular Biology, Vol. 684, 2011, which is hereby incorporated hereinby reference. Recombination between pJB 161 and the endogenous plasmidsin vivo yield engineered microbes expressing the genes of interest fromtheir endogenous plasmids. Alternatively, vectors can be engineered torecombine with the host cell chromosome, or the vector can be engineeredto replicate and express genes of interest independent of the host cellchromosome or any of the host cell's endogenous plasmids.

A further example of a vector suitable for recombinant proteinproduction is the pET system (Novagen®). This system has beenextensively characterized for use in E. coli and other microorganisms.In this system, target genes are cloned in pET plasmids under control ofstrong bacteriophage T7 transcription and (optionally) translationsignals; expression is induced by providing a source of T7 RNApolymerase in the host cell. T7 RNA polymerase is so selective andactive that, when fully induced, almost all of the microorganism'sresources are converted to target gene expression; the desired productcan comprise more than 50% of the total cell protein a few hours afterinduction. It is also possible to attenuate the expression level simplyby lowering the concentration of inducer. Decreasing the expressionlevel may enhance the soluble yield of some target proteins. In someembodiments this system also allows for maintenance of target genes in atranscriptionally silent un-induced state.

In some embodiments of using this system, target genes are cloned usinghosts that do not contain the T7 RNA polymerase gene, thus alleviatingpotential problems related to plasmid instability due to the productionof proteins potentially toxic to the host cell. Once established in anon-expression host, target protein expression may be initiated eitherby infecting the host with λCE6, a phage that carries the T7 RNApolymerase gene under the control of the λ pL and pI promoters, or bytransferring the plasmid into an expression host containing achromosomal copy of the T7 RNA polymerase gene under lacUV5 control. Inthe second case, expression is induced by the addition of IPTG orlactose to the bacterial culture or using an autoinduction medium. Otherplasmids systems that are controlled by the lac operator, but do notrequire the T7 RNA polymerase gene and rely upon E. coli's native RNApolymerase include the pTrc plasmid suite (Invitrogen) or pQE plamidsuite (QIAGEN).

In other embodiments it is possible to clone directly into expressionhosts. Two types of T7 promoters and several hosts that differ in theirstringency of suppressing basal expression levels are available,providing great flexibility and the ability to optimize the expressionof a wide variety of target genes.

Suitable vectors for expression of nucleic acids in mammalian cellstypically comprise control functions provided by viral regulatoryelements. For example, commonly used promoters are derived from polyomavirus, Adenovirus 2, cytomegalovirus, or Simian Virus 40.

D. Promoters

Promoters useful for expressing the recombinant genes described hereininclude both constitutive and inducible/repressible promoters. Examplesof inducible/repressible promoters include nickel-inducible promoters(e.g., PnrsA, PnrsB; see, e.g., Lopez-Mauy et al., Cell (2002) v.43:247-256) and urea repressible promoters such as PnirA (described in,e.g., Qi et al., Applied and Environmental Microbiology (2005) v.71:5678-5684). Additional examples of inducible/repressible promotersinclude PnirA (promoter that drives expression of the nirA gene, inducedby nitrate and repressed by urea) and Psuf (promoter that drivesexpression of the sufB gene, induced by iron stress). Examples ofconstitutive promoters include Pcpc (promoter that drives expression ofthe cpc operon), Prbc (promoter that drives expression of rubisco),PpsbAII (promoter that drives expression of PpsbAII), Pcro (lambda phagepromoter that drives expression of cro). In other embodiments, a PaphI1and/or a laclq-Ptrc promoter can used to control expression. Wheremultiple recombinant genes are expressed in an engineered microorganism,the different genes can be controlled by different promoters or byidentical promoters in separate operons, or the expression of two ormore genes may be controlled by a single promoter as part of an operon.

Further non-limiting examples of inducible promoters may include, butare not limited to, those induced by expression of an exogenous protein(e.g., T7 RNA polymerase, SP6 RNA polymerase), by the presence of asmall molecule (e.g., IPTG, galactose, tetracycline, steroid hormone,abscisic acid), by absence of small molecules (e.g., CO₂, iron,nitrogen), by metals or metal ions (e.g., copper, zinc, cadmium,nickel), and by environmental factors (e.g., heat, cold, stress, light,darkness), and by growth phase. In some embodiments, the induciblepromoter is tightly regulated such that in the absence of induction,substantially no transcription is initiated through the promoter. Insome embodiments, induction of the promoter does not substantially altertranscription through other promoters. Also, generally speaking, thecompound or condition that induces an inducible promoter is not benaturally present in the organism or environment where expression issought.

In some embodiments, the inducible promoter is induced by limitation ofCO₂ supply to a cyanobacteria culture. By way of non-limiting example,the inducible promoter may be the promoter sequence of Synechocystis PCC6803 that are up-regulated under the CO₂-limitation conditions, such asthe cmp genes, ntp genes, ndh genes, sbt genes, chp genes, and rbcgenes, or a variant or fragment thereof.

In some embodiments, the inducible promoter is induced by ironstarvation or by entering the stationary growth phase. In someembodiments, the inducible promoter may be variant sequences of thepromoter sequence of cyanobacterial genes that are up-regulated underFe-starvation conditions such as isiA, or when the culture enters thestationary growth phase, such as isiA, phrA, sigC, sigB, and sigH genes,or a variant or fragment thereof.

In some embodiments, the inducible promoter is induced by a metal ormetal ion. By way of non-limiting example, the inducible promoter may beinduced by copper, zinc, cadmium, mercury, nickel, gold, silver, cobalt,and bismuth or ions thereof. In some embodiments, the inducible promoteris induced by nickel or a nickel ion. In some embodiments, the induciblepromoter is induced by a nickel ion, such as Ni²⁺. In another exemplaryembodiment, the inducible promoter is the nickel inducible promoter fromSynechocystis PCC 6803. In another embodiment, the inducible promotermay be induced by copper or a copper ion. In yet another embodiment, theinducible promoter may be induced by zinc or a zinc ion. In stillanother embodiment, the inducible promoter may be induced by cadmium ora cadmium ion. In yet still another embodiment, the inducible promotermay be induced by mercury or a mercury ion. In an alternativeembodiment, the inducible promoter may be induced by gold or a gold ion.In another alternative embodiment, the inducible promoter may be inducedby silver or a silver ion. In yet another alternative embodiment, theinducible promoter may be induced by cobalt or a cobalt ion. In stillanother alternative embodiment, the inducible promoter may be induced bybismuth or a bismuth ion.

In some embodiments, the promoter is induced by exposing a cellcomprising the inducible promoter to a metal or metal ion. The cell maybe exposed to the metal or metal ion by adding the metal to themicrobial growth media. In certain embodiments, the metal or metal ionadded to the microbial growth media may be efficiently recovered fromthe media. In other embodiments, the metal or metal ion remaining in themedia after recovery does not substantially impede downstream processingof the media or of the bacterial gene products.

Further non-limiting examples of constitutive promoters includeconstitutive promoters from Gram-negative bacteria or a bacteriophagepropagating in a Gram-negative bacterium. For instance, promoters forgenes encoding highly expressed Gram-negative gene products may be used,such as the promoter for Lpp, OmpA, rRNA, and ribosomal proteins.Alternatively, regulatable promoters may be used in a strain that lacksthe regulatory protein for that promoter. For instance P_(lac), P_(tac),and P_(trc), may be used as constitutive promoters in strains that lackLac1. Similarly, P22 P_(R) and P_(L) may be used in strains that lackthe lambda C2 repressor protein, and lambda P_(R) and P_(L) may be usedin strains that lack the lambda C1 repressor protein. In one embodiment,the constitutive promoter is from a bacteriophage. In anotherembodiment, the constitutive promoter is from a Salmonellabacteriophage. In yet another embodiment, the constitutive promoter isfrom a cyanophage. In some embodiments, the constitutive promoter is aSynechocystis promoter. For instance, the constitutive promoter may bethe PpsbAll promoter or its variant sequences, the Prbc promoter or itsvariant sequences, the P_(cpc) promoter or its variant sequences, andthe PrnpB promoter or its variant sequences.

E. Host Cells

Also provided are host cells transformed with the nucleic acid moleculesor vectors disclosed herein, and descendants thereof. In someembodiments the host cells are microbial cells. In some embodiments, thehost cells carry the nucleic acid sequences on vectors, which may butneed not be freely replicating vectors. In other embodiments, thenucleic acids have been integrated into the genome of the host cellsand/or into an endogenous plasmid of the host cells. The transformedhost cells find use, e.g., in the production of recombinant nutritiveproteins disclosed herein.

“Microorganisms” includes prokaryotic and eukaryotic microbial speciesfrom the Domains Archaea, Bacteria and Eucarya, the latter includingyeast and filamentous fungi, protozoa, algae, or higher Protista. Theterms “microbial cells” and “microbes” are used interchangeably with theterm microorganism.

A variety of host microorganisms may be transformed with a nucleic acidsequence disclosed herein and may in some embodiments be used to producea recombinant nutritive protein disclosed herein. Suitable hostmicroorganisms include both autotrophic and heterotrophic microbes. Insome applications the autotrophic microorganisms allows for a reductionin the fossil fuel and/or electricity inputs required to make anutritive protein encoded by a recombinant nucleic acid sequenceintroduced into the host microorganism. This, in turn, in someapplications reduces the cost and/or the environmental impact ofproducing the nutritive protein and/or reduces the cost and/or theenvironmental impact in comparison to the cost and/or environmentalimpact of manufacturing alternative nutritive proteins, such as whey,egg, and soy. For example, the cost and/or environmental impact ofmaking a nutritive protein disclosed herein using a host microorganismas disclosed herein is in some embodiments lower that the cost and/orenvironmental impact of making whey protein in a form suitable for humanconsumption by processing of cows milk.

Non-limiting examples of heterotrophs include Escherichia coli,Salmonella typhimurium, Bacillus subtilis, Bacillus megaterium,Corynebacterium glutamicum, Streptomyces coelicolor, Streptomyceslividans, Streptomyces vanezuelae, Streptomyces roseosporus,Streptomyces fradiae, Streptomyces griseus, Streptomyces calvuligerus,Streptomyces hygroscopicus, Streptomyces platensis, Saccharopolysporaerythraea, Corynebacterium glutamicum, Aspergillus niger, Aspergillusnidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus sojae,Penicillium chrysogenum, Trichoderma reesei, Clostridium acetobutylicum,Clostridium beijerinckii, Clostridium thermocellum, Fusibacterpaucivorans, Saccharomyces cerevisiae, Saccharomyces boulardii, Pichiapastoris, and Pichia stipitis.

Photoautotrophic microorganisms include eukaryotic algae, as well asprokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfurbacteria, purple sulfur bacteria, and purple non-sulfur bacteria.

Extremophiles are also contemplated as suitable organisms. Suchorganisms withstand various environmental parameters such astemperature, radiation, pressure, gravity, vacuum, desiccation,salinity, pH, oxygen tension, and chemicals. They includehyperthermophiles, which grow at or above 80° C. such as Pyrolobusfumarii; thermophiles, which grow between 60-80° C. such asSynechococcus lividis; mesophiles, which grow between 15-60° C.; andpsychrophiles, which grow at or below 15° C. such as Psychrobacter andsome insects. Radiation tolerant organisms include Deinococcusradiodurans. Pressure-tolerant organisms include piezophiles, whichtolerate pressure of 130 MPa. Weight-tolerant organisms includebarophiles. Hypergravity (e.g., >1 g) hypogravity (e.g., <1 g) tolerantorganisms are also contemplated. Vacuum tolerant organisms includetardigrades, insects, microbes and seeds. Dessicant tolerant andanhydrobiotic organisms include xerophiles such as Artemia salina;nematodes, microbes, fungi and lichens. Salt-tolerant organisms includehalophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina.pH-tolerant organisms include alkaliphiles such as Natronobacterium,Bacillus firmus OF4, Spirulina spp. (e.g., pH>9) and acidophiles such asCyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, whichcannot tolerate O₂ such as Methanococcus jannaschii; microaerophils,which tolerate some O₂ such as Clostridium and aerobes, which require O₂are also contemplated. Gas-tolerant organisms, which tolerate pure CO₂include Cyanidium caldarium and metal tolerant organisms includemetalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn),Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life onthe Edge: Amazing Creatures Thriving in Extreme Environments. New York:Plenum (1998) and Seckbach, J. “Search for Life in the Universe withTerrestrial Microbes Which Thrive Under Extreme Conditions.” InCristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds.,Astronomical and Biochemical Origins and the Search for Life in theUniverse, p. 511. Milan: Editrice Compositori (1997).

Mixotrophic organisms are also suitable organisms. Mixotrophic organismscan utilize a mix of different sources of energy and carbon, forexample, photo- and chemotrophy, litho- and organotrophy, auto- andheterotrophy, and combinations thereof or a combination of it.Mixotrophs can be either eukaryotic or prokaryotic. Additionally,mixotrophs can be obligate or facultative. Suitable mixotrophicorganisms include mixotrophic algae and mixotrophic bacteria.

Algae and cyanobacteria include but are not limited to the followinggenera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes,Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium,Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora,Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus,Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa,Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus,Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella,Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys,Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis,Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira,Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis,Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena,Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros,Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis,Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris,Characiopsis, Characium, Charales, Chilomonas, Chlainomonas,Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis,Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella,Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea,Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema,Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton,Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas,Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa,Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus,Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta,Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera,Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium,Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,Diplostauron, Distrionella, Docidium, Drapamaldia, Dunaliella,Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis,Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis,Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta,Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia,Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta,Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis,Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax,Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia,Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga,Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea,Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium,Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia,Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema,Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium,Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne,Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron,Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium,Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia,Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis,Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella,Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira,Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias,Microchaete, Microcoleus, Microcystis, Microglena, Micromonas,Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus,Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris,Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium,Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema,Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria,Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus,Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas,Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium,Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium,Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis,Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora,Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema,Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus,Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra,Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis,Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella,Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus,Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma,Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium,Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis,Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas,Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma,Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spennatozopsis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus,Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella,Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium,Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella,Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix,Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria,Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella,Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria,Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.

Additional cyanobacteria include members of the genus Chamaesiphon,Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis,Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus,Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella,Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira,Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya,Microcoleus, Oscillatoria, Planktothrix, Prochiorothrix, Pseudanabaena,Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena,Anabaenopsis, Aphanizomenon, Cyanospira, Cylindrospermopsis,Cylindrospermum, Nodularia, Nostoc, Scylonema, Calothrix, Rivularia,Tolypothrix, Chlorogloeopsis, Fischerella, Geitieria, Iyengariella,Nostochopsis, Stigonema and Thermosynechococcus.

Green non-sulfur bacteria include but are not limited to the followinggenera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix,Herpetosiphon, Roseiflexus, and Thermomicrobium.

Green sulfur bacteria include but are not limited to the followinggenera: Chlorobium, Clathrochloris, and Prosthecochloris.

Purple sulfur bacteria include but are not limited to the followinggenera: Allochromatium, Chromatium, Halochromatium, Isochromatium,Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa,Thiorhodococcus, and Thiocystis.

Purple non-sulfur bacteria include but are not limited to the followinggenera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium,Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum,Rodovibrio, and Roseospira.

Aerobic chemolithotrophic bacteria include but are not limited tonitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp.,Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp.,Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibriosp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligatelychemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., ironand manganese-oxidizing and/or depositing bacteria such as Siderococcussp., and magnetotactic bacteria such as Aquaspirillum sp.

Archaeobacteria include but are not limited to methanogenicarchaeobacteria such as Methanobacterium sp., Methanobrevibacter sp.,Methanothermus sp., Methanococcus sp., Methanomicrobium sp.,Methanospirillum sp., Methanogenium sp., Methanosarcina sp.,Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanussp.; extremely thermophilic S-Metabolizers such as Thermoproteus sp.,Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganismssuch as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp.,Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp.,Mycobacteria sp., and oleaginous yeast.

Yet other suitable organisms include synthetic cells or cells producedby synthetic genomes as described in Venter et al. US Pat. Pub. No.2007/0264688, and cell-like systems or synthetic cells as described inGlass et al. US Pat. Pub. No. 2007/0269862.

Still other suitable organisms include Escherichia coli, Acetobacteraceti, Bacillus subtilis, yeast and fungi such as Clostridiumljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pseudomonas fluorescens, or Zymomonas mobilis. In some embodiments thoseorganisms are engineered to fix carbon dioxide while in otherembodiments they are not.

In some embodiments eukaryotic cells, such as insect cells or mammaliancells, such as human cells are used as host cells. Vectors andexpression control sequences including promoters and enhancers are wellknown for such cells. Examples of useful mammalian host cell lines forthis purpose are monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

F. Production of Nutritive Proteins

Skilled artisans are aware of many suitable methods available forculturing recombinant cells to produce (and optionally secrete) arecombinant nutritive protein as disclosed herein, as well as forpurification and/or isolation of expressed recombinant proteins. Themethods chosen for protein purification depend on many variables,including the properties of the protein of interest, its location andform within the cell, the vector, host strain background, and theintended application for the expressed protein. Culture conditions canalso have an effect on solubility and localization of a given targetprotein. Many approaches can be used to purify target proteins expressedin recombinant microbial cells as disclosed herein, including withoutlimitation ion exchange and gel filtration.

In some embodiments a peptide fusion tag is added to the recombinantprotein making possible a variety of affinity purification methods thattake advantage of the peptide fusion tag. In some embodiments, the useof an affinity method enables the purification of the target protein tonear homogeneity in one step. Purification may include cleavage of partor all of the fusion tag with enterokinase, factor Xa, thrombin, or HRV3C proteases, for example. In some embodiments, before purification oractivity measurements of an expressed target protein, preliminaryanalysis of expression levels, cellular localization, and solubility ofthe target protein is performed. The target protein may be found in anyor all of the following fractions: soluble or insoluble cytoplasmicfractions, periplasm, or medium. Depending on the intended application,preferential localization to inclusion bodies, medium, or theperiplasmic space can be advantageous, in some embodiments, for rapidpurification by relatively simple procedures.

While Escherichia coli is widely regarded as a robust host forheterologous protein expression, it is also widely known thatover-expression of many proteins in this host is prone to aggregation inthe form of insoluble inclusion bodies. One of the most commonly usedmethods for either rescuing inclusion body formation, or to improve thetiter of the protein itself, is to include an amino-terminalmaltose-binding protein (MBP) [Austin B P, Nallamsetty S, Waugh D S.Hexahistidine-tagged maltose-binding protein as a fusion partner for theproduction of soluble recombinant proteins in Escherichia coli. MethodsMol Biol. 2009; 498:157-72], or small ubiquitin-related modifier (SUMO)[Saitoh H, Uwada J, Azusa K. Strategies for the expression ofSUMO-modified target proteins in Escherichia coli. Methods Mol Biol.2009; 497:211-21; Malakhov M P, Mattern M R, Malakhova O A, Drinker M,Weeks S D, Butt T R. SUMO fusions and SUMO-specific protease forefficient expression and purification of proteins. J Struct FunctGenomics. 2004; 5(1-2):75-86; Panavas T, Sanders C, Butt T R. SUMOfusion technology for enhanced protein production in prokaryotic andeukaryotic expression systems. Methods Mol Biol. 2009; 497:303-17]fusion to the protein of interest. These two proteins are expressedextremely well, and in the soluble form, in Escherichia coli such thatthe protein of interest is also effectively produced in the solubleform. The protein of interest can be cleaved by designing a sitespecific protease recognition sequence (such as the tobacco etch virus(TEV) protease) in-between the protein of interest and the fusionprotein [1].

In some embodiments the recombinant protein is initially not foldedcorrectly or is insoluble. A variety of methods are well known forrefolding of insoluble proteins. Most protocols comprise the isolationof insoluble inclusion bodies by centrifugation followed bysolubilization under denaturing conditions. The protein is then dialyzedor diluted into a non-denaturing buffer where refolding occurs. Becauseevery protein possesses unique folding properties, the optimal refoldingprotocol for any given protein can be empirically determined by askilled artisan. Optimal refolding conditions can, for example, berapidly determined on a small scale by a matrix approach, in whichvariables such as protein concentration, reducing agent, redoxtreatment, divalent cations, etc., are tested. Once the optimalconcentrations are found, they can be applied to a larger scalesolubilization and refolding of the target protein.

In some embodiments the nutritive protein does not comprise a tertiarystructure. In some embodiments less than half of the amino acids in thenutritive protein participate in a tertiary structure. In someembodiments the nutritive protein does not comprise a secondarystructure. In some embodiments less than half of the amino acids in thenutritive protein participate in a secondary structure. Recombinantnutritive proteins may be isolated from a culture of cells expressingthem in a state that comprises one or more of these structural features.In some embodiments the tertiary structure of a recombinant nutritiveprotein is reduced or eliminated after the protein is isolated from aculture producing it. In some embodiments the secondary structure of arecombinant nutritive protein is reduced or eliminated after the proteinis isolated from a culture producing it.

In some embodiments a CAPS buffer at alkaline pH in combination withN-lauroylsarcosine is used to achieve solubility of the inclusionbodies, followed by dialysis in the presence of DTT to promoterefolding. Depending on the target protein, expression conditions, andintended application, proteins solubilized from washed inclusion bodiesmay be >90% homogeneous and may not require further purification.Purification under fully denaturing conditions (before refolding) ispossible using His•Tag® fusion proteins and His•Bind® immobilized metalaffinity chromatography (Novogen®). In addition, S•Tag™ T7•Tag®, andStrep•Tag® II fusion proteins solubilized from inclusion bodies using 6M urea can be purified under partially denaturing conditions by dilutionto 2 M urea (S•Tag and T7-Tag) or 1 M urea (Strep•Tag II) prior tochromatography on the appropriate resin. Refolded fusion proteins can beaffinity purified under native conditions using His•Tag, S•Tag,Strep•Tag II, and other appropriate affinity tags (e.g., GST•Tag™, andT7•Tag) (Novogen®).

In some embodiments the recombinant nutritive protein is an endogenousprotein of the host cell used to express it. That is, the cellulargenome of the host cell comprises an open reading frame that encodes therecombinant nutritive protein. In some embodiments regulatory sequencessufficient to increase expression of the nutritive protein are insertedinto the host cell genome and operatively linked to the endogenous openreading frame such that the regulatory sequences drive overexpression ofthe recombinant nutritive protein from a recombinant nucleic acid. Insome embodiments heterologous nucleic acid sequences are fused to theendogenous open reading frame of the nutritive protein and cause thenutritive protein to be synthesized comprising a heterologous amino acidsequence that changes the cellular trafficking of the recombinantnutritive protein, such as directing it to an organelle or to asecretion pathway. In some embodiments an open reading frame thatencodes the endogeneous host cell protein is introduced into the hostcell on a plasmid that further comprises regulatory sequencesoperatively linked to the open reading frame. In some embodiments therecombinant host cell expresses at least 2 times, at least 3 times, atleast 4 times, at least 5 times, at least 10 times, or at least 20times, at least 30 times, at least 40 times, at least 50 times, or atleast 100 times more of the recombinant nutritive protein than theamount of the nutritive protein produced by a similar host cell grownunder similar conditions.

In some embodiments nutritive proteins of this disclosure aresynthesized chemically without the use of a recombinant productionsystem. Protein synthesis can be carried out in a liquid-phase system orin a solid-phase system using techniques known in the art (see, e.g.,Atherton, E., Sheppard, R. C. (1989). Solid Phase peptide synthesis: apractical approach. Oxford, England: IRL Press; Stewart, J. M., Young,J. D. (1984). Solid phase peptide synthesis (2nd ed.). Rockford: PierceChemical Company.

G. Production of Recombinant Nutritive Proteins in Plants

The nucleic acid molecules comprising a nucleic acid sequence encoding anutritive protein of this disclosure enable production of transgenicplants comprising the nucleic acid sequence. Accordingly, thisdisclosure also provides plant comprising a recombinant nucleic acidmolecule comprising a nucleic acid sequence encoding a nutritive proteinof this disclosure. The plant can be any plant that is subject totransformation and regeneration and include, but are not limited to,Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus,avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli,brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya,cauliflower, celery, Chinese cabbage, cherry, cilantro, citrus,clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant,endive, escarole, eucalyptus, fennel, figs, forest trees, gourd, grape,grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime,Loblolly pine, mango, melon, mushroom, nut, oat, okra, onion, orange, anornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry,rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry,sugarbeet, sugarcane, sunflower, sweet corn, sweet potato, sweetgum,tangerine, tea, tobacco, tomato, turf, a vine, watermelon, wheat, yams,and zucchini. In preferred embodiments, the plant is a bean, broccoli,cabbage, canola, carrot, cauliflower, celery, Chinese cabbage, corn,cotton cucumber, eggplant, leek, lettuce, melon, pea, pepper, pumpkin,radish, spinach, soybean, squash, sugarcane, sweet corn, tomato,watermelon, and wheat plant. In some embodiments, the plant is a cornplant. In some embodiments, the plant is a soybean plant. In someembodiments, the plant is a cotton plant. In some embodiments, the plantis a canola plant. In some embodiments the plant is a member of a genusselected from Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum,Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus. For instance, see U.S. Pat. Nos. 5,858,742and 5,322,938, which disclose versions of the constitutive promoterderived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No.5,641,876, which discloses a rice actin promoter, U.S. PatentApplication Publication 2002/0192813A1, which discloses 5′, 3′ andintron elements useful in the design of effective plant expressionvectors, U.S. patent application Ser. No. 09/757,089, which discloses amaize chloroplast aldolase promoter, U.S. patent application Ser. No.08/706,946, which discloses a rice glutelin promoter, U.S. patentapplication Ser. No. 09/757,089, which discloses a maize aldolase (FDA)promoter, and U.S. patent application Ser. No. 60/310,370, whichdiscloses a maize nicotianamine synthase promoter. These and numerousother promoters that function in plant cells are known to those skilledin the art and available for use in recombinant nucleic acids to providefor expression of nutritive proteins in transgenic plants.

For some applications preferential expression in plant green tissues isdesired. Promoters of interest for such uses include those from genessuch as Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase(Rubisco) small subunit (Fischhoff et al. (1992) Plant Mol. Biol.20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK)(Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42-48).

Furthermore, the promoters may be altered to contain at least oneenhancer sequence to assist in elevating gene expression. Such enhancersare known in the art. By including an enhancer sequence with suchconstructs, the expression of the nutritive protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedupstream (5′) or downstream (3′) to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Particularly usefulas enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No.5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase geneintron, the maize heat shock protein 70 gene intron (U.S. Pat. No.5,593,874) and the maize shrunken 1 gene.

For some applications expression in plant seed tissues is desired toeffect modify seed composition. Exemplary promoters for use for seedcomposition modification include promoters from seed genes such as napin(U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252),zein Z27 (Russell et al. (1997) Transgenic Res. 6(2):157-166), globulin1 (Belanger et al (1991) Genetics 129:863-872), glutelin 1 (Russell(1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy et al. (1996)Plant Mol. Biol. 31(6):1205-1216)

Recombinant nucleic acid constructs prepared in accordance with thedisclosure will also generally include a 3′ element that typicallycontains a polyadenylation signal and site. Well-known 3′ elementsinclude those from Agrobacterium tumefaciens genes such as nos 3′, tml3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat.No. 6,090,627; 3′ elements from plant genes such as wheat (Triticumaesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, awheat fructose-1,6-biphosphatase gene, a rice glutelin gene a ricelactate dehydrogenase gene and a rice beta-tubulin gene, all of whichare disclosed in U.S. published patent application 2002/0192813 A1; andthe pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′),and 3′ elements from the genes within the host plant.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For descriptions of the use ofchloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat.No. 5,728,925. For description of the transit peptide region of anArabidopsis EPSPS gene, see Klee, H. J. et al (MGG (1987) 210:437-442).

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present disclosure. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat) and Agrobacterium-mediated transformation is described in U.S.Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S.Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean). ForAgrobacterium tumefaciens based plant transformation system, additionalelements present on transformation constructs will include T-DNA leftand right border sequences to facilitate incorporation of therecombinant polynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function in plants, including cre-loxas disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695.

Transformation methods are generally practiced in tissue culture onmedia and in a controlled environment. “Media” refers to the numerousnutrient mixtures that are used to grow cells in vitro, that is, outsideof the intact living organism. Recipient cell targets include, but arenot limited to, meristem cells, callus, immature embryos and gameticcells such as microspores, pollen, sperm and egg cells. It iscontemplated that any cell from which a fertile plant may be regeneratedis useful as a recipient cell. Callus may be initiated from tissuesources including, but not limited to, immature embryos, seedling apicalmeristems, microspores and the like. Cells capable of proliferating ascallus are also recipient cells for genetic transformation. Practicaltransformation methods and materials for making transgenic plants ofthis disclosure, for example various media and recipient target cells,transformation of immature embryo cells and subsequent regeneration offertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and6,232,526.

The seeds of transgenic plants can be harvested from fertile transgenicplants and used to grow progeny generations of transformed plants thatproduce the recombinant nutritive protein of this disclosure. Inaddition to direct transformation of a plant with a recombinant DNA,transgenic plants can be prepared by crossing a first plant having arecombinant DNA with a second plant lacking the DNA. For example,recombinant DNA can be introduced into first plant line that is amenableto transformation to produce a transgenic plant which can be crossedwith a second plant line to introgress the recombinant DNA into thesecond plant line. A transgenic plant with recombinant DNA encoding anutritive protein of this disclosure, can be crossed with transgenicplant line having other recombinant DNA that confers another trait, forexample herbicide resistance or pest resistance, or production of asecond nutritive product such as an oil, to produce progeny plantshaving recombinant DNA that confers both traits. Typically, in suchbreeding for combining traits the transgenic plant donating theadditional trait is a male line and the transgenic plant carrying thebase traits is the female line. The progeny of this cross will segregatesuch that some of the plants will carry the DNA for both parental traitsand some will carry DNA for one parental trait; such plants can beidentified by markers associated with parental recombinant DNA, e.g.marker identification by analysis for recombinant DNA or, in the casewhere a selectable marker is linked to the recombinant, by applicationof the selecting agent such as a herbicide for use with a herbicidetolerance marker, or by selection for the enhanced trait. Progeny plantscarrying DNA for both parental traits can be crossed back into thefemale parent line multiple times, for example usually 6 to 8generations, to produce a progeny plant with substantially the samegenotype as one original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theherbicides to which the transformed plants may be resistant are usefulagents for selective markers. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(aroA or EPSPS). Examples of such selectable are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Selectablemarkers which provide an ability to visually identify transformants canalso be employed, for example, a gene expressing a colored orfluorescent protein such as a luciferase or green fluorescent protein(GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) forwhich various chromogenic substrates are known.

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets regenerated from transformed plant cells can be transferred toplant growth mix, and hardened off, for example, in an environmentallycontrolled chamber. Plants are regenerated from about 6 weeks to 10months after a transformant is identified, depending on the initialtissue. Plants may be pollinated using conventional plant breedingmethods known to those of skill in the art and seed produced, forexample self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA and selected for thepresence of a heterologous nutritive protein.

Transgenic plants derived from the plant cells of this disclosure aregrown to generate transgenic plants comprising the heterologous nucleicacid that encodes a nutritive protein of this disclosure and producetransgenic seed and haploid pollen comprising the heterologous nucleicacid sequence. Such plants with enhanced traits are identified byselection of transformed plants or progeny seed for the enhanced trait.Transgenic plants grown from transgenic seed provided herein demonstrateimproved agronomic traits that contribute to increased yield or othertraits that provide increased plant value, including, for example,improved protein quality such as increasing the content of at least oneof essential amino acids, branch chain amino acids, or Leu.

The transgenic plants are useful as sources of nutritive proteins. Forexample, in some embodiments a transgenic plant comprising a recombinantnutritive protein of this disclosure comprises an increased weightfraction of total protein compared to a control non-transgenic plant. Insome embodiments a transgenic plant comprising a recombinant nutritiveprotein of this disclosure comprises an increased weight fraction ofessential amino acids compared to a control non-transgenic plant. Insome embodiments a transgenic plant comprising a recombinant nutritiveprotein of this disclosure comprises an increased weight fraction ofbranch chain amino acids compared to a control non-transgenic plant. Insome embodiments a transgenic plant comprising a recombinant nutritiveprotein of this disclosure comprises an increased weight fraction of Leucompared to a control non-transgenic plant. In some embodiments atransgenic plant comprising a recombinant nutritive protein of thisdisclosure comprises at least one of: a) an increased ratio of branchchain amino acid residues to total amino acid residues compared to acontrol non-transgenic plant; b) an increased ratio of Leu residues tototal amino acid residues compared to a control non-transgenic plant;and c) an increased ratio of essential amino acid residues to totalamino acid residues compared to a control non-transgenic plant. In someembodiments a transgenic plant comprising a recombinant nutritiveprotein of this disclosure comprises: a) an increased ratio of branchchain amino acid residues to total amino acid residues compared to acontrol non-transgenic plant; b) an increased ratio of Leu residues tototal amino acid residues compared to a control non-transgenic plant;and c) an increased ratio of essential amino acid residues to totalamino acid residues compared to a control non-transgenic plant.

Accordingly, the transgenic plants are useful as sources of high qualityprotein. The plants may be harvested and used in mammalian diets with orwithout further processing. For example, flour made from transgenicwheat, cornmeal made from transgenic corn, or rice or rice flour derivedfrom transgenic rice is enriched in at least one of protein, essentialamino acids, branch chain amino acids, and Leu compared to similarproducts made from plants that do not comprise the recombinant nutritiveprotein. In some embodiments the recombinant nutritive protein is aplant protein or comprises a polypeptide sequence of a plant protein ora derivative or mutein thereof, such as but not necessarily a protein orpolypeptide sequence of the same type of plant. In other embodiments therecombinant nutritive protein is not a plant protein or a derivative ormutein thereof.

In some embodiments the recombinant nutritive protein is recovered orpartially recovered from the transgenic plant before it is consumed by amammal.

H. Compositions

At least one nutritive protein disclosed herein may be combined with atleast one second component to form a nutritive composition. In someembodiments the only source of amino acid in the composition is the atleast one nutritive protein disclosed herein. In such embodiments theamino acid composition of the composition will be the same as the aminoacid composition of the at least one nutritive protein disclosed herein.In some embodiments the composition comprises at least one nutritiveprotein disclosed herein and at least one second protein. In someembodiments the at least one second protein is a second nutritiveprotein disclosed herein, while in other embodiments the at least onesecond protein is not a nutritive protein disclosed herein. In someembodiments the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more nutritive proteins disclosedherein. In some embodiments the composition comprises 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more proteinsthat are not nutritive proteins disclosed herein. In some embodimentsthe composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more nutritive proteins and the compositioncomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more proteins that are not nutritive proteins disclosedherein.

In some embodiments the nutritive composition as described in thepreceding paragraph, further comprises at least one of at least onepolypeptide, at least one peptide, and at least one free amino acid. Insome embodiments the nutritive composition comprises at least onepolypeptide and at least one peptide. In some embodiments the nutritivecomposition comprises at least one polypeptide and at least one freeamino acid. In some embodiments the nutritive composition comprises atleast one peptide and at least one free amino acid. In some embodimentsthe at least one polypeptide, at least one peptide, and/or at least onefree amino acid comprises amino acids selected from 1) branch chainamino acids, 2) leucine, and 3) essential amino acids. In someembodiments the at least one polypeptide, at least one peptide, and/orat least one free amino acid consists of amino acids selected from 1)branch chain amino acids, 2) leucine, and 3) essential amino acids. Insome embodiments, the nutritive composition comprises at least onemodified amino acid or a non-standard amino acid. Modified amino acidsinclude amino acids that have modifications to one or more of thecarboxy terminus, amino terminus, and/or side chain. Non-standard aminoacids may be selected from those that are formed by post-translationalmodification of proteins, for example, carboxylated glutamate,hydroxyproline, or hypusine. Other non-standard amino acids are notfound in proteins. Examples include lanthionine, 2-aminoisobutyric acid,dehydroalanine, gamma-aminobutyric acid, ornithine and citrulline. Insome embodiments, the nutritive composition comprises one or moreD-amino acids. In some embodiments, the nutritive composition comprisesone or more L-amino acids. In some embodiments, the nutritivecomposition comprises a mixture of one or more D-amino acids and one ormore L-amino acids.

By adding at least one of a polypeptide, a peptide, and a free aminoacid to a nutritive composition the proportion of at least one of branchchain amino acids, leucine, and essential amino acids, to total aminoacid, present in the composition can be increased.

In some embodiments the composition comprises at least one carbohydrate.A “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide”may be used interchangeably. Most carbohydrates are aldehydes or ketoneswith many hydroxyl groups, usually one on each carbon atom of themolecule. Carbohydrates generally have the molecular formula CnH2nOn. Acarbohydrate may be a monosaccharide, a disaccharide, trisaccharide,oligosaccharide, or polysaccharide. The most basic carbohydrate is amonosaccharide, such as glucose, sucrose, galactose, mannose, ribose,arabinose, xylose, and fructose. Disaccharides are two joinedmonosaccharides. Exemplary disaccharides include sucrose, maltose,cellobiose, and lactose. Typically, an oligosaccharide includes betweenthree and six monosaccharide units (e.g., raffinose, stachyose), andpolysaccharides include six or more monosaccharide units. Exemplarypolysaccharides include starch, glycogen, and cellulose. Carbohydratesmay contain modified saccharide units such as 2′-deoxyribose wherein ahydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group isreplace with a fluorine, or N-acetylglucosamine, a nitrogen-containingform of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose).Carbohydrates may exist in many different forms, for example,conformers, cyclic forms, acyclic forms, stereoisomers, tautomers,anomers, and isomers.

In some embodiments the composition comprises at least one lipid. Asused herein a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids may be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

In some embodiments the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments the composition comprises at least one supplementalvitamin. The at least one vitamin can be fat-soluble or water solublevitamins. Suitable vitamins include but are not limited to vitamin C,vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin,vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenicacid, and biotin. Suitable forms of any of the foregoing are salts ofthe vitamin, derivatives of the vitamin, compounds having the same orsimilar activity of the vitamin, and metabolites of the vitamin.

In some embodiments the composition comprises at least one organism.Suitable examples are well known in the art and include probiotics(e.g., species of Lactobacillus or Bifidobacterium), spirulina,chlorella, and porphyra.

In some embodiments the composition comprises at least one dietarysupplement. Suitable examples are well known in the art and includeherbs, botanicals, and certain hormones. Non limiting examples includeginko, gensing, and melatonin.

In some embodiments the composition comprises an excipient. Non-limitingexamples of suitable excipients include a buffering agent, apreservative, a stabilizer, a binder, a compaction agent, a lubricant, adispersion enhancer, a disintegration agent, a flavoring agent, asweetener, a coloring agent.

In some embodiments the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments the excipient comprises a preservative. Non-limitingexamples of suitable preservatives include antioxidants, such asalpha-tocopherol and ascorbate, and antimicrobials, such as parabens,chlorobutanol, and phenol.

In some embodiments the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate, isomorphoussilicate, and microcrystalline cellulose as high HLB emulsifiersurfactants.

In some embodiments the composition comprises a disintegrant as anexcipient. In some embodiments the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-crystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pecitin, andtragacanth. In some embodiments the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments the excipient comprises a flavoring agent. Flavoringagents incorporated into the outer layer can be chosen from syntheticflavor oils and flavoring aromatics; natural oils; extracts from plants,leaves, flowers, and fruits; and combinations thereof. In someembodiments the flavoring agent is selected from cinnamon oils; oil ofwintergreen; peppermint oils; clover oil; hay oil; anise oil;eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape andgrapefruit oil; and fruit essences including apple, peach, pear,strawberry, raspberry, cherry, plum, pineapple, and apricot.

In some embodiments the excipient comprises a sweetener. Non-limitingexamples of suitable sweeteners include glucose (corn syrup), dextrose,invert sugar, fructose, and mixtures thereof (when not used as acarrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; and sugar alcohols such as sorbitol,mannitol, sylitol, and the like. Also contemplated are hydrogenatedstarch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In some embodiments the composition comprises a coloring agent.Non-limiting examples of suitable color agents include food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), and externaldrug and cosmetic colors (Ext. D&C). The coloring agents can be used asdyes or their corresponding lakes.

The weight fraction of the excipient or combination of excipients in theformulation is usually about 50% or less, about 45% or less, about 40%or less, about 35% or less, about 30% or less, about 25% or less, about20% or less, about 15% or less, about 10% or less, about 5% or less,about 2% or less, or about 1% or less of the total weight of the aminoacids in the composition.

The nutritive proteins and nutritive compositions disclosed herein canbe formulated into a variety of forms and administered by a number ofdifferent means. The compositions can be administered orally, rectally,or parenterally, in formulations containing conventionally acceptablecarriers, adjuvants, and vehicles as desired. The term “parenteral” asused herein includes subcutaneous, intravenous, intramuscular, orintrasternal injection and infusion techniques. In an exemplaryembodiment, the nutritive protein or composition is administered orally.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, troches, lozenges, powders, and granules. A capsuletypically comprises a core material comprising a nutritive protein orcomposition and a shell wall that encapsulates the core material. Insome embodiments the core material comprises at least one of a solid, aliquid, and an emulsion. In some embodiments the shell wall materialcomprises at least one of a soft gelatin, a hard gelatin, and a polymer.Suitable polymers include, but are not limited to: cellulosic polymerssuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose (HPMC), methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulosesuccinate and carboxymethylcellulose sodium; acrylic acid polymers andcopolymers, such as those formed from acrylic acid, methacrylic acid,methyl acrylate, ammonio methylacrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate (e.g., those copolymers soldunder the trade name “Eudragit”); vinyl polymers and copolymers such aspolyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate,vinylacetate crotonic acid copolymer, and ethylene-vinyl acetatecopolymers; and shellac (purified lac). In some embodiments at least onepolymer functions as taste-masking agents.

Tablets, pills, and the like can be compressed, multiply compressed,multiply layered, and/or coated. The coating can be single or multiple.In one embodiment, the coating material comprises at least one of asaccharide, a polysaccharide, and glycoproteins extracted from at leastone of a plant, a fungus, and a microbe. Non-limiting examples includecorn starch, wheat starch, potato starch, tapioca starch, cellulose,hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin,mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gumkaraya, gum ghatti, tragacanth gum, funori, carrageenans, agar,alginates, chitosans, or gellan gum. In some embodiments the coatingmaterial comprises a protein. In some embodiments the coating materialcomprises at least one of a fat and an oil. In some embodiments the atleast one of a fat and an oil is high temperature melting. In someembodiments the at least one of a fat and an oil is hydrogenated orpartially hydrogenated. In some embodiments the at least one of a fatand an oil is derived from a plant. In some embodiments the at least oneof a fat and an oil comprises at least one of glycerides, free fattyacids, and fatty acid esters. In some embodiments the coating materialcomprises at least one edible wax. The edible wax can be derived fromanimals, insects, or plants. Non-limiting examples include beeswax,lanolin, bayberry wax, carnauba wax, and rice bran wax. Tablets andpills can additionally be prepared with enteric coatings.

Alternatively, powders or granules embodying the nutritive proteins andnutritive compositions disclosed herein can be incorporated into a foodproduct. In some embodiments the food product is be a drink for oraladministration. Non-limiting examples of a suitable drink include fruitjuice, a fruit drink, an artificially flavored drink, an artificiallysweetened drink, a carbonated beverage, a sports drink, a liquid diaryproduct, a shake, an alcoholic beverage, a caffeinated beverage, infantformula and so forth. Other suitable means for oral administrationinclude aqueous and nonaqueous solutions, creams, pastes, emulsions,suspensions and slurries, each of which may optionally also contain atleast one of suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, coloring agents, and flavoringagents.

In some embodiments the food product is a solid foodstuff. Suitableexamples of a solid foodstuff include without limitation a food bar, asnack bar, a cookie, a brownie, a muffin, a cracker, a biscuit, a creamor paste, an ice cream bar, a frozen yogurt bar, and the like.

In some embodiments, the nutritive proteins and nutritive compositionsdisclosed herein are incorporated into a therapeutic food. In someembodiments, the therapeutic food is a ready-to-use food that optionallycontains some or all essential macronutrients and micronutrients. Insome embodiments, the nutritive proteins and nutritive compositionsdisclosed herein are incorporated into a supplementary food that isdesigned to be blended into an existing meal. In some embodiments, thesupplemental food contains some or all essential macronutrients andmicronutrients. In some embodiments, the nutritive proteins andnutritive compositions disclosed herein are blended with or added to anexisting food to fortify the food's protein nutrition. Examples includefood staples (grain, salt, sugar, cooking oil, margarine), beverages(coffee, tea, soda, beer, liquor, sports drinks), snacks, sweets andother foods.

The compositions disclosed herein can be utilized in methods to increaseat least one of muscle mass, strength and physical function,thermogenesis, metabolic expenditure, satiety, mitochondrial biogenesis,weight or fat loss, and lean body composition for example.

I. Methods of Use

In some embodiments the nutritive proteins and nutritive compositionsdisclosed herein are administered to a patient or a user (sometimescollectively referred to as a “subject”). As used herein “administer”and “administration” encompasses embodiments in which one person directsanother to consume a nutritive protein or nutritive composition in acertain manner and/or for a certain purpose, and also situations inwhich a user uses a nutritive protein or nutritive composition in acertain manner and/or for a certain purpose independently of or invariance to any instructions received from a second person. Non-limitingexamples of embodiments in which one person directs another to consume anutritive protein or nutritive composition in a certain manner and/orfor a certain purpose include when a physician prescribes a course ofconduct and/or treatment to a patient, when a trainer advises a user(such as an athlete) to follow a particular course of conduct and/ortreatment, and when a manufacturer, distributer, or marketer recommendsconditions of use to an end user, for example through advertisements orlabeling on packaging or on other materials provided in association withthe sale or marketing of a product.

In some embodiments the nutritive proteins or nutritive compositions areprovided in a dosage form. In some embodiments the dosage form isdesigned for administration of at least one nutritive protein disclosedherein, wherein the total amount of nutritive protein administered isselected from 0.1 g to 1 g, 1 g to 5 g, from 2 g to 10 g, from 5 g to 15g, from 10 g to 20 g, from 15 g to 25 g, from 20 g to 40 g, from 25-50g, and from 30-60 g. In some embodiments the dosage form is designed foradministration of at least one nutritive protein disclosed herein,wherein the total amount of nutritive protein administered is selectedfrom about 0.1 g, 0.1 g-1 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9g, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g, 65g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, and 100 g.

In some embodiments the dosage form is designed for administration of atleast one nutritive protein disclosed herein, wherein the total amountof essential amino acids administered is selected from 0.1 g to 1 g,from 1 g to 5 g, from 2 g to 10 g, from 5 g to 15 g, from 10 g to 20 g,and from 1-30 g. In some embodiments the dosage form is designed foradministration of at least one nutritive protein disclosed herein,wherein the total amount of nutritive protein administered is selectedfrom about 0.1 g, 0.1-1 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g,10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g, 65 g,70 g, 75 g, 80 g, 85 g, 90 g, 95 g, and 100 g.

In some embodiments the nutritive protein or nutritive composition isconsumed at a rate of from 0.1 g to 1 g a day, 1 g to 5 g a day, from 2g to 10 g a day, from 5 g to 15 g a day, from 10 g to 20 g a day, from15 g to 30 g a day, from 20 g to 40 g a day, from 25 g to 50 g a day,from 40 g to 80 g a day, from 50 g to 100 g a day, or more.

In some embodiments, of the total protein intake by the subject, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or about 100% ofthe total protein intake by the subject over a dietary period is made upof at least one nutritive protein according to this disclosure. In someembodiments, of the total protein intake by the subject, from 5% to 100%of the total protein intake by the subject, from 5% to 90% of the totalprotein intake by the subject, from 5% to 80% of the total proteinintake by the subject, from 5% to 70% of the total protein intake by thesubject, from 5% to 60% of the total protein intake by the subject, from5% to 50% of the total protein intake by the subject, from 5% to 40% ofthe total protein intake by the subject, from 5% to 30% of the totalprotein intake by the subject, from 5% to 20% of the total proteinintake by the subject, from 5% to 10% of the total protein intake by thesubject, from 10% to 100% of the total protein intake by the subject,from 10% to 100% of the total protein intake by the subject, from 20% to100% of the total protein intake by the subject, from 30% to 100% of thetotal protein intake by the subject, from 40% to 100% of the totalprotein intake by the subject, from 50% to 100% of the total proteinintake by the subject, from 60% to 100% of the total protein intake bythe subject, from 70% to 100% of the total protein intake by thesubject, from 80% to 100% of the total protein intake by the subject, orfrom 90% to 100% of the total protein intake by the subject, over adietary period, is made up of at least one nutritive protein accordingto this disclosure. In some embodiments the at least one nutritiveprotein of this disclosure accounts for at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50% of the subject's calorie intakeover a dietary period.

In some embodiments the at least one nutritive protein according to thisdisclosure comprises at least 2 nutritive proteins of this disclosure,at least 3 nutritive proteins of this disclosure, at least 4 nutritiveproteins of this disclosure, at least 5 nutritive proteins of thisdisclosure, at least 6 nutritive proteins of this disclosure, at least 7nutritive proteins of this disclosure, at least 8 nutritive proteins ofthis disclosure, at least 9 nutritive proteins of this disclosure, atleast 10 nutritive proteins of this disclosure, or more.

In some embodiments the dietary period is 1 meal, 2 meals, 3 meals, atleast 1 day, at least 2 days, at least 3 days, at least 4 days, at least5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months, or atleast 1 year. In some embodiments the dietary period is from 1 day to 1week, from 1 week to 4 weeks, from 1 month, to 3 months, from 3 monthsto 6 months, or from 6 months to 1 year.

Clinical studies provide evidence that protein prevents muscle loss dueto aging or bed rest. In particular, studies have shown that proteinsupplementation increases muscle fractional synthetic rate (FSR) duringprolonged bed rest, maintains leg mass and strength during prolonged bedrest, increases lean body mass, improves functional measures of gait andbalance, and may serve as a viable intervention for individuals at riskof sarcopenia due to immobility or prolonged bed rest. (See, e.g.,Paddon-Jones D, et al. J Clin Endocrinol Metab 2004, 89:4351-4358;Ferrando, A et al. Clinical Nutrition 2009 1-6; Katsanos C et al. Am JPhysiol Endocrinol Metab. 2006, 291: 381-387).

Studies on increasing muscle protein anabolism in athletes have shownthat protein provided following exercise promotes muscle hypertrophy toa greater extent than that achieved by exercise alone. It has also beenshown that protein provided following exercise supports proteinsynthesis without any increase in protein breakdown, resulting in a netpositive protein balance and muscle mass accretion. While muscle proteinsynthesis appears to respond in a dose-response fashion to essentialamino acid supplementation, not all proteins are equal in buildingmuscle. For example, the amino acid leucine is an important factor instimulating muscle protein synthesis. (See, e.g., Borscheim E et al. AmJ Physiol Endocrinol Metab 2002, 283: E648-E657; Borsheim E et al. ClinNutr. 2008, 27: 189-95; Esmarck B et al J Physiol 2001, 535: 301-311;Moore D et al. Am J Clin Nutr 2009, 89: 161-8).

In another aspect this disclosure provides methods of maintaining orincreasing at least one of muscle mass, muscle strength, and functionalperformance in a subject. In some embodiments the methods compriseproviding to the subject a sufficient amount of a nutritive protein ofthis disclosure, a nutritive composition of this disclosure, or anutritive composition made by a method of this disclosure. In someembodiments the subject is at least one of elderly, critically-medicallyill, and suffering from protein-energy malnutrition. In some embodimentsthe sufficient amount of a nutritive protein of this disclosure, anutritive composition of this disclosure, or a nutritive compositionmade by a method of this disclosure is consumed by the subject incoordination with performance of exercise. In some embodiments thenutritive protein of this disclosure, nutritive composition of thisdisclosure, or nutritive composition made by a method of this disclosureis consumed by the subject by an oral, enteral, or parenteral route.

In another aspect this disclosure provides methods of maintaining orachieving a desirable body mass index in a subject. In some embodimentsthe methods comprise providing to the subject a sufficient amount of anutritive protein of this disclosure, a nutritive composition of thisdisclosure, or a nutritive composition made by a method of thisdisclosure. In some embodiments the subject is at least one of elderly,critically-medically ill, and suffering from protein-energymalnutrition. In some embodiments the sufficient amount of a nutritiveprotein of this disclosure, a nutritive composition of this disclosure,or a nutritive composition made by a method of this disclosure isconsumed by the subject in coordination with performance of exercise. Insome embodiments the nutritive protein of this disclosure, nutritivecomposition of this disclosure, or nutritive composition made by amethod of this disclosure is consumed by the subject by an oral,enteral, or parenteral route.

In another aspect this disclosure provides methods of providing proteinto a subject with protein-energy malnutrition. In some embodiments themethods comprise providing to the subject a sufficient amount of anutritive protein of this disclosure, a nutritive composition of thisdisclosure, or a nutritive composition made by a method of thisdisclosure. In some embodiments the nutritive protein of thisdisclosure, nutritive composition of this disclosure, or nutritivecomposition made by a method of this disclosure is consumed by thesubject by an oral, enteral, or parenteral route.

The need for essential amino acid supplementation has been suggested incancer patients and other patients suffering from cachexia. Dietarystudies in mice have shown survival and functional benefits to cachecticcancer-bearing mice through dietary intervention with essential aminoacids. Beyond cancer, essential amino acid supplementation has alsoshown benefits, such as improved muscle function and muscle gain, inpatients suffering from other diseases who have difficulty exercisingand therefore suffer from muscular deterioration, such as chronicobstructive pulmonary disease, chronic heart failure, HIV, and otherdisease states.

Studies have shown that specific amino acids have advantages in managingcachexia. A relatively high content of BCAAs and Leu in diets arethought to have a positive effect in cachexia by promoting total proteinsynthesis by signaling an increase in translation, enhancing insulinrelease, and inhibiting protein degradation. Thus, consuming increaseddietary BCAAs in general and/or Leu in particular will contributepositively to reduce or reverse the effects of cachexia. Becausenitrogen balance is important in countering the underlying cause ofcachexia it is thought that consuming increased dietary glutamine and/orarginine will contribute positively to reduce or reverse the effects ofcachexia. (See, e.g., Op den Kamp C, Langen R, Haegens A, Schols A.“Muscle atrophy in cachexia: can dietary protein tip the balance?”Current Opinion in Clinical Nutrition and Metabolic Care 2009,12:611-616; Poon R T-P, Yu W-C, Fan S-T, et al. “Long-term oral branchedchain amino acids in patients undergoing chemoembolization forhepatocellular carcinoma: a randomized trial.” Aliment Pharmacol Ther2004; 19:779-788; Tayek J A, Bistrian B R, Hehir D J, Martin R, MoldawerL L, Blackburn G L. “Improved protein kinetics and albumin synthesis bybranched chain amino acid-enriched total parenteral nutrition in cancercachexia.” Cancer. 1986; 58:147-57; Xi P, Jiang Z, Zheng C, Lin Y, Wu G“Regulation of protein metabolism by glutamine: implications fornutrition and health.” Front Biosci. 2011 Jan. 1; 16:578-97).

Accordingly, also provided herein are methods of treating cachexia in asubject. In some embodiments a sufficient amount of a nutritive proteinof this disclosure, a nutritive composition of this disclosure, or anutritive composition made by a method of this disclosure for a subjectwith cachexia is an amount such that the amount of protein of thisdisclosure ingested by the person meets or exceeds the metabolic needs(which are often elevated). A protein intake of 1.5 g/kg of body weightper day or 15-20% of total caloric intake appears to be an appropriatetarget for persons with cachexia. In some embodiments all of the proteinconsumed by the subject is a nutritive protein according to thisdisclosure. In some embodiments nutritive protein according to thisdisclosure is combined with other sources of protein and/or free aminoacids to provide the total protein intake of the subject. In someembodiments the subject is at least one of elderly, critically-medicallyill, and suffering from protein-energy malnutrition. In some embodimentsthe subject suffers from a disease that makes exercise difficult andtherefore causes muscular deterioration, such as chronic obstructivepulmonary disease, chronic heart failure, HIV, cancer, and other diseasestates. In some embodiments, the nutritive protein according todisclosure, the nutritive composition according to disclosure, or thenutritive composition made by a method according to disclosure isconsumed by the subject in coordination with performance of exercise. Insome embodiments, the nutritive protein according to this disclosure,the nutritive composition according to disclosure, or the nutritivecomposition made by a method according to disclosure is consumed by thesubject by an oral, enteral, or parenteral route.

Sarcopenia is the degenerative loss of skeletal muscle mass (typically0.5-1% loss per year after the age of 25), quality, and strengthassociated with aging. Sarcopenia is a component of the frailtysyndrome. The European Working Group on Sarcopenia in Older People(EWGSOP) has developed a practical clinical definition and consensusdiagnostic criteria for age-related sarcopenia. For the diagnosis ofsarcopenia, the working group has proposed using the presence of bothlow muscle mass and low muscle function (strength or performance).Sarcopenia is characterized first by a muscle atrophy (a decrease in thesize of the muscle), along with a reduction in muscle tissue “quality,”caused by such factors as replacement of muscle fibres with fat, anincrease in fibrosis, changes in muscle metabolism, oxidative stress,and degeneration of the neuromuscular junction. Combined, these changeslead to progressive loss of muscle function and eventually to frailty.Frailty is a common geriatric syndrome that embodies an elevated risk ofcatastrophic declines in health and function among older adults.Contributors to frailty can include sarcopenia, osteoporosis, and muscleweakness. Muscle weakness, also known as muscle fatigue, (or “lack ofstrength”) refers to the inability to exert force with one's skeletalmuscles. Weakness often follows muscle atrophy and a decrease inactivity, such as after a long bout of bedrest as a result of anillness. There is also a gradual onset of muscle weakness as a result ofsarcopenia.

The nutritive proteins of this disclosure are useful for treatingsarcopenia or frailty once it develops in a subject or for preventingthe onset of sarcopenia or frailty in a subject who is a member of an atrisk groups. In some embodiments all of the protein consumed by thesubject is a nutritive protein according to this disclosure. In someembodiments nutritive protein according to this disclosure is combinedwith other sources of protein and/or free amino acids to provide thetotal protein intake of the subject. In some embodiments the subject isat least one of elderly, critically-medically ill, and suffering fromprotein-energy malnutrition. In some embodiments, the nutritive proteinaccording to disclosure, the nutritive composition according todisclosure, or the nutritive composition made by a method according todisclosure is consumed by the subject in coordination with performanceof exercise. In some embodiments, the nutritive protein according tothis disclosure, the nutritive composition according to disclosure, orthe nutritive composition made by a method according to disclosure isconsumed by the subject by an oral, enteral, or parenteral route.

Obesity is a multifactoral disorder associated with a host ofcomorbidities including hypertension, type 2 diabetes, dyslipidemia,coronary heart disease, stroke, cancer (eg, endometrial, breast, andcolon), osteoarthritis, sleep apnea, and respiratory problems. Theincidence of obesity, defined as a body mass index >30 kg/m2, hasincreased dramatically in the United States, from 15% (1976-1980) to 33%(2003-2004), and it continues to grow. Although the mechanismscontributing to obesity are complex and involve the interplay ofbehavioral components with hormonal, genetic, and metabolic processes,obesity is largely viewed as a lifestyle-dependent condition with 2primary causes: excessive energy intake and insufficient physicalactivity. With respect to energy intake, there is evidence that modestlyincreasing the proportion of protein in the diet, while controllingtotal energy intake, may improve body composition, facilitate fat loss,and improve body weight maintenance after weight loss. Positive outcomesassociated with increased dietary protein are thought to be dueprimarily to lower energy intake associated with increased satiety,reduced energy efficiency and/or increased thermogenesis, positiveeffects on body composition (specifically lean muscle mass), andenhanced glycemic control.

Dietary proteins are more effective in increasing post-prandial energyexpenditure than isocaloric intakes of carbohydrates or fat (see, e.g.,Dauncey M, Bingham S. “Dependence of 24 h energy expenditure in man oncomposition of the nutrient intake.” Br J Nutr 1983, 50: 1-13; Karst Het al. “Diet-induced thermogenesis in man: thermic effects of singleproteins, carbohydrates and fats depending on their energy amount.” AnnNutr Metab. 1984, 28: 245-52; Tappy L et al “Thermic effect of infusedamino acids in healthy humans and in subjects with insulin resistance.”Am J Clin Nutr 1993, 57 (6): 912-6). This property along with otherproperties (satiety induction; preservation of lean body mass) makeprotein an attractive component of diets directed at weight management.The increase in energy expenditure caused by such diets may in part bedue to the fact that the energy cost of digesting and metabolizingprotein is higher than for other calorie sources. Protein turnover,including protein synthesis, is an energy consuming process. Inaddition, high protein diets may also up-regulate uncoupling protein inliver and brown adipose, which is positively correlated with increasesin energy expenditure. It has been theorized that different proteins mayhave unique effects on energy expenditure.

Studies suggest that ingestion of protein, particularly proteins withhigh EAA and/or BCAA content, leads to distinct effects on thermogenesisand energy expenditure (see, e.g., Mikkelsen P. et al. “Effect offat-reduced diets on 24 h energy expenditure: comparisons between animalprotein, vegetable protein and carbohydrate.” Am J Clin Nutr 2000,72:1135-41; Acheson K. et al. “Protein choices targeting thermogenesisand metabolism.” Am J Clin Nutr 2011, 93:525-34; Alfenas R. et al.“Effects of protein quality on appetite and energy metabolism in normalweight subjects” Arg Bras Endocrinol Metabol 2010, 54 (1): 45-51;Lorenzen J. et al. “The effect of milk proteins on appetite regulationand diet-induced thermogenesis.” J Clin Nutr 2012 66 (5): 622-7).Additionally, L-tyrosine has been identified as an amino acid that playsa role in thermogenesis (see, e.g., Belza A. et al. “The beta-adrenergicantagonist propranolol partly abolishes thermogenic response tobioactive food ingredients.” Metabolism 2009, 58 (8):1137-44). Furtherstudies suggest that Leucine and Arginine supplementation appear toalter energy metabolism by directing substrate to lean body mass ratherthan adipose tissue (Dulloo A. “The search for compounds that stimulatethermogenesis in obesity management: from pharmaceuticals to functionalfood ingredients.” Obes Rev 2011 12: 866-83).

Collectively the literature suggests that different protein types leadsto distinct effects on thermogenesis. Because proteins or peptides richin EAAs, BCAA, and/or at least one of Tyr, Arg, and Leu are believed tohave a stimulatory effect on thermogenesis, and because stimulation ofthermogenesis is believed to lead to positive effects on weightmanagement, this disclosure also provides products and methods useful tostimulation thermogenesis and/or to bring about positive effects onweight management in general.

More particularly, this disclosure provides methods of increasingthermogenesis in a subject. In some embodiments the methods compriseproviding to the subject a sufficient amount of a nutritive protein ofthis disclosure, a nutritive composition of this disclosure, or anutritive composition made by a method of this disclosure. In someembodiments the subject is obese. In some embodiments, the nutritiveprotein according to disclosure, the nutritive composition according todisclosure, or the nutritive composition made by a method according todisclosure is consumed by the subject in coordination with performanceof exercise. In some embodiments, the nutritive protein according todisclosure, the nutritive composition according to disclosure, or thenutritive composition made by a method according to disclosure isconsumed by the subject by an oral, enteral, or parenteral route.

At the basic level, the reason for the development of an overweightcondition is due to an imbalance between energy intake and energyexpenditure. Attempts to reduce food at any particular occasion(satiation) and across eating occasions (satiety) have been a majorfocus of recent research. Reduced caloric intake as a consequence offeeling satisfied during a meal and feeling full after a meal resultsfrom a complex interaction of internal and external signals. Variousnutritional studies have demonstrated that variation in food propertiessuch as energy density, content, texture and taste influence bothsatiation and satiety.

There are three macronutrients that deliver energy: fat, carbohydratesand proteins. A gram of protein or carbohydrate provides 4 calorieswhile a gram of fat 9 calories. Protein generally increases satiety to agreater extent than carbohydrates or fat and therefore may facilitate areduction in calorie intake. However, there is considerable evidencethat indicates the type of protein matters in inducing satiety (see,e.g., W. L. Hall, et al. “Casein and whey exert different effects onplasma amino acid profiles, gastrointestinal hormone secretion andappetite.” Br J Nutr. 2003 February, 89(2):239-48; R. Abou-Samra, et al.“Effect of different protein sources on satiation and short-term satietywhen consumed as a starter.” Nutr J. 2011 Dec. 23, 10:139; T. Akhavan,et al. “Effect of premeal consumption of whey protein and itshydrolysate on food intake and postmeal glycemia and insulin responsesin young adults.” Am J Clin Nutr. 2010 April, 91(4):966-75, Epub 2010Feb. 17; MA Veldhorst “Dose-dependent satiating effect of whey relativeto casein or soy” Physiol Behav. 2009 Mar. 23, 96(4-5):675-82). Evidenceindicates that protein rich in Leucine is particularly effective atinducing satiety (see, e.g., Fromentin G et al “Peripheral and centralmechanisms involved in the control of food intake by dietary amino acidsand proteins.” Nutr Res Rev 2012 25: 29-39).

In some embodiments a nutritive protein of this disclosure is consumedby a subject concurrently with at least one pharmaceutical or biologicdrug product. In some embodiments the beneficial effects of thenutritive protein and the at least one pharmaceutical or biologic drugproduct have an additive effect while in some embodiments the beneficialeffects of the nutritive protein and the at least one pharmaceutical orbiologic drug product have a synergistic effect. Examples ofpharmaceutical or biologic drug products that can be administered withthe nutritive proteins of this disclosure are well known in the art. Forexample, when a nutritive protein of this disclosure is used to maintainor increase at least one of muscle mass, muscle strength, and functionalperformance in a subject, the nutritive protein may be consumed by asubject concurrently with a therapeutic dosage regime of at least onepharmaceutical or biologic drug product indicated to maintain orincrease at least one of muscle mass, muscle strength, and functionalperformance in a subject, such as an anabolic steroid. When a nutritiveprotein of this disclosure is used to maintain or achieve a desirablebody mass index in a subject, the nutritive protein may be consumed by asubject concurrently with a therapeutic dosage regime of at least onepharmaceutical or biologic drug product indicated to maintain or achievea desirable body mass index in a subject, such as orlistat, lorcaserin,sibutramine, rimonabant, metformin, exenatide, or pramlintide. When anutritive protein of this disclosure is used to induce at least one of asatiation response and a satiety response in a subject, the nutritiveprotein may be consumed by a subject concurrently with a therapeuticdosage regime of at least one pharmaceutical or biologic drug productindicated to induce at least one of a satiation response and a satietyresponse in a subject, such as rimonabant, exenatide, or pramlintide.When a nutritive protein of this disclosure is used to treat at leastone of cachexia, sarcopenia and frailty in a subject, the nutritiveprotein may be consumed by a subject concurrently with a therapeuticdosage regime of at least one pharmaceutical or biologic drug productindicated to treat at least one of cachexia, sarcopenia and frailty,such as omega-3 fatty acids or anabolic steroids. Because of the role ofdietary protein in inducing satiation and satiety, the nutritiveproteins and nutritive compositions disclosed herein can be used toinduce at least one of a satiation response and a satiety response in asubject. In some embodiments the methods comprise providing to thesubject a sufficient amount of a nutritive protein of this disclosure, anutritive composition of this disclosure, or a nutritive compositionmade by a method of this disclosure. In some embodiments the subject isobese. In some embodiments, the nutritive protein according todisclosure, the nutritive composition according to disclosure, or thenutritive composition made by a method according to disclosure isconsumed by the subject in coordination with performance of exercise. Insome embodiments, the nutritive protein according to disclosure, thenutritive composition according to disclosure, or the nutritivecomposition made by a method according to disclosure is consumed by thesubject by an oral, enteral, or parenteral route.

In some embodiments incorporating a least one nutritive protein ornutritive composition of this disclosure into the diet of a subject hasat least one effect selected from inducing postprandial satiety(including by suppressing hunger), inducing thermogenesis, reducingglycemic response, positively affecting energy expenditure positivelyaffecting lean body mass, reducing the weight gain caused by overeating,and decreasing energy intake. In some embodiments incorporating a leastone nutritive protein or nutritive composition of this disclosure intothe diet of a subject has at least one effect selected from increasingloss of body fat, reducing lean tissue loss, improving lipid profile,and improving glucose tolerance and insulin sensitivity in the subject.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe invention. These examples are presented for illustrative purposesand should not serve to limit the true scope of the invention.

Example 1: Identification of Protein Fragments Containing Ratios ofEssential Amino Acids, Branch Chain Amino Acids, and Leucine Greaterthan or Equal to Whey and Containing all Essential Amino Acids

The UniProtKB/Swiss-Prot (a collaboration between the EuropeanBioinformatics Institute and the Swiss Institute of Bioinformatics) is amanually curated and reviewed protein database, and was used as thestarting point for protein identification. Proteins from the ediblespecies Solanum lycopersicum, Zea mays, Oryza sativa subsp. Japonica,Glycine max, Ovis aries, Pisum sativum, Spinacia oleracea, Oryza sativasubsp. Indica, Triticum aestivum, Sus scrofa, Prunus persica, Capsicumannuum, Malus domestica, Thunnus albacares, Capra hircus, Cicerarietinum, Salmo salar, Meleagris gallopavo, Solanum tuberosum, andAgaricus bisporus having greater than or equal to fifty (50) amino acidswere considered as viable targets. Roughly 8,415 such proteins wereselected for evaluation and the proteins were then fragmented intoroughly fifty (50), roughly one hundred (100), or roughly one hundredand fifty (150) amino acids portions of the original protein, designedto contain digestive enzyme cleavage sites on both ends. In addition,the proteins were screened against a database of known allergens todetermine whether any had greater than 50% global homology to a knownallergen. A total of 1,000 fragments were identified that containgreater than or equal to 51% EAA, greater than or equal to 25% BCAA, andgreater than or equal to 13% Leu, and contain all essential amino acids,and have less than 50% global homology to known allergens (SEQ ID NOS: 1to 1000). (Note that for Examples 1-5, 51% EAA/25% BCAA/13% Leurepresent values that are each 1-2% greater than those of whey (definedherein to be 49% EAA/24% BCAA/11% Leu). These values were used toidentify nutritive protein fragments of interest for the purposes ofthis Example only, in order to ensure that the identified proteins havea higher content of EAA, BCAA, and Leu than whey). For the set ofproteins the solvation score at pH 7 (“SolvScore”), aggregation score atpH 7 (“AggScore”), allergenicity (i.e., percent local homology to knownallergens, as described herein), toxicity (i.e., percent homology toknown toxins, as described herein), anti-nutricity (i.e., percenthomology to known protease inhibitors, as described herein), and humanhomology (i.e., percent homology to known human proteins, as describedherein) were calculated, and the total number of Cys residues (“C”) weredetermined. The characteristics of 75 representative fragments thusidentified are presented in Tables 3A and 3B.

TABLE 3A Seq ID No UniProt FragEnds EAA BCAA L C 1 Q29550 101:301 0.530.27 0.13 3 2 Q29078  1:176 0.52 0.27 0.14 5 3 Q29550 201:426 0.56 0.250.13 2 4 Q29078  1:76 0.60 0.34 0.20 3 5 Q9N2D1  1:76 0.57 0.27 0.18 3 6Q29079  1:76 0.56 0.29 0.18 1 7 Q29550 101:176 0.53 0.31 0.15 1 8 Q70BM6201:326 0.58 0.26 0.15 0 9 Q70BM6 201:426 0.59 0.30 0.17 3 10 Q70BM6101:326 0.56 0.28 0.14 2 11 O81122 101:301 0.53 0.29 0.16 1 12 Q9M7M1101:201 0.55 0.28 0.19 1 13 O81122 101:201 0.55 0.28 0.19 1 14 Q9M7M1101:301 0.53 0.28 0.16 1 15 Q8WNV7 151:226 0.51 0.29 0.14 1 16 P50118176:276 0.52 0.26 0.15 3 17 P12263 451:526 0.55 0.26 0.13 0 18 Q29197 26:126 0.52 0.29 0.18 0 19 O02799 151:301 0.54 0.26 0.19 2 20 P80229201:276 0.55 0.27 0.18 1 21 Q29624 226:326 0.59 0.25 0.13 2 22 A5GFQ5 26:151 0.52 0.25 0.15 3 23 Q5GN48 1601:1676 0.57 0.26 0.15 0 24 Q5GN481026:1101 0.55 0.26 0.16 2 25 A6M931 176:276 0.54 0.26 0.13 1 26 P49039 1:101 0.51 0.27 0.14 0 27 P79430 226:326 0.62 0.25 0.14 3 28 P61291 1:151 0.53 0.27 0.14 2 29 B5X2B8 126:326 0.52 0.25 0.13 3 30 Q1A730226:301 0.52 0.28 0.20 2 31 D2SW95 276:476 0.52 0.31 0.13 3 32 O02799251:326 0.57 0.29 0.22 1 33 B5RI54 126:251 0.54 0.26 0.13 2 34 P21999 76:176 0.51 0.28 0.13 5 35 Q07511 151:276 0.52 0.27 0.13 2 36 A5A779351:476 0.53 0.31 0.24 4 37 Q6ITT4 476:601 0.54 0.30 0.16 1 38 O46374 876:1076 0.55 0.26 0.13 2 39 Q4U116  901:1076 0.55 0.25 0.14 2 40P04045 376:476 0.53 0.27 0.13 1 41 Q29121 151:226 0.52 0.30 0.13 2 42Q2VEC3 1601:1726 0.54 0.27 0.16 1 43 B5X737  26:176 0.57 0.26 0.13 2 44Q9XSW2 201:401 0.53 0.25 0.15 8 45 P19756 276:351 0.60 0.32 0.14 0 46P67931 101:201 0.51 0.26 0.19 1 47 Q9AWA5 401:476 0.58 0.27 0.13 0 48O46374  976:1051 0.56 0.26 0.19 2 49 Q9XSW2 301:401 0.59 0.29 0.16 3 50Q8MIQ9 551:651 0.56 0.28 0.15 1 51 O02668 526:626 0.53 0.26 0.14 0 52Q6UAQ8  51:226 0.51 0.27 0.13 3 53 P21343 326:401 0.54 0.33 0.17 1 54P79430 126:326 0.58 0.26 0.13 5 55 P10691  26:176 0.52 0.25 0.14 0 56O02697 626:701 0.52 0.27 0.19 1 57 Q5VI41 676:751 0.53 0.30 0.13 1 58O97716 201:376 0.52 0.25 0.13 4 59 Q66UW5  51:151 0.52 0.25 0.16 2 60D2SW95  1:201 0.54 0.27 0.14 5 61 O46491 201:351 0.53 0.25 0.15 1 62Q52QT8  1:151 0.53 0.26 0.16 5 63 Q9P4T7  1:76 0.53 0.25 0.17 0 64Q2VEE8 201:276 0.60 0.25 0.14 0 65 P37831 251:376 0.52 0.26 0.16 0 66B9EPI1  1:126 0.56 0.30 0.16 4 67 B5X2B8 126:201 0.61 0.30 0.15 0 68O02799 526:651 0.57 0.26 0.16 1 69 Q99028  76:151 0.53 0.30 0.16 3 70Q07511  51:276 0.51 0.26 0.13 4 71 P08454  1:76 0.57 0.28 0.13 1 72Q5GN48 2251:2501 0.51 0.26 0.14 0 73 O64390 276:426 0.52 0.25 0.15 1 74P09954  1:101 0.53 0.31 0.14 0 75 P29196 801:876 0.57 0.28 0.18 0

TABLE 3B Seq ID No SolvScore AggScore Allergenicity ToxicityAntinutricity Human Homology 1 −16.14 0.70 0.58 0.40 0.23 0.83 2 −15.720.57 0.54 0.00 0.25 0.50 3 −18.83 0.61 0.45 0.30 0.00 0.76 4 −18.32 0.800.43 0.28 0.29 0.46 5 −10.78 0.78 0.42 0.42 0.29 0.78 6 −18.03 0.62 0.410.28 0.28 0.48 7 −14.86 0.78 0.40 0.43 0.26 0.79 8 −17.96 0.60 0.36 0.000.25 0.81 9 −13.70 0.90 0.36 0.00 0.21 0.82 10 −15.03 0.80 0.36 0.000.22 0.81 11 −18.93 0.53 0.36 0.00 0.21 0.00 12 −22.36 0.47 0.35 0.210.00 0.00 13 −22.36 0.47 0.35 0.21 0.00 0.00 14 −18.90 0.52 0.35 0.000.24 0.00 15 −12.53 0.69 0.35 0.00 0.00 0.82 16 −20.41 0.43 0.35 0.200.25 0.86 17 −18.65 0.43 0.25 0.43 0.24 0.83 18 −28.11 0.37 0.31 0.000.00 0.98 19 −26.99 0.37 0.33 0.24 0.22 0.81 20 −26.56 0.35 0.34 0.291.00 0.79 21 −26.33 0.75 0.30 0.29 0.22 0.84 22 −25.32 0.42 0.29 0.230.29 0.95 23 −25.11 0.34 0.29 0.00 0.00 0.93 24 −24.91 0.25 0.28 0.000.26 0.88 25 −24.78 0.47 0.31 0.00 0.20 1.00 26 −24.60 0.44 0.29 0.250.31 0.25 27 −24.55 0.79 0.28 0.24 0.27 0.77 28 −24.41 0.38 0.33 0.000.25 1.00 29 −24.09 0.41 0.31 0.00 0.25 0.50 30 −23.74 0.38 0.27 0.260.26 0.99 31 −23.68 0.62 0.31 0.00 0.23 1.00 32 −23.68 0.38 0.27 0.000.24 0.80 33 −23.57 0.39 0.32 0.00 0.00 0.89 34 −23.51 0.64 0.33 0.170.27 0.99 35 −23.41 0.45 0.34 0.21 0.00 0.29 36 −23.38 0.50 0.32 0.230.26 0.98 37 −23.11 0.63 0.30 0.22 0.24 1.00 38 −23.11 0.41 0.33 0.220.00 0.99 39 −23.02 0.72 0.29 0.00 0.23 0.99 40 −22.98 0.36 0.30 0.000.28 0.57 41 −22.88 0.59 0.30 0.27 0.29 1.00 42 −22.87 0.49 0.32 0.000.23 0.00 43 −22.79 0.42 0.31 0.21 0.23 0.59 44 −22.74 0.56 0.31 0.000.21 0.88 45 −22.73 0.80 0.26 0.00 0.30 0.91 46 −22.72 0.50 0.29 0.000.30 0.73 47 −22.62 0.38 0.23 0.23 0.00 0.24 48 −22.55 0.42 0.24 0.270.24 1.00 49 −22.53 0.71 0.31 0.00 0.26 0.96 50 −22.43 0.59 0.30 0.000.00 0.90 51 −22.38 0.48 0.30 0.00 1.00 0.76 52 −22.32 0.47 0.33 0.000.00 0.96 53 −22.21 0.49 0.26 0.00 0.26 0.30 54 −22.18 0.64 0.31 0.200.21 0.78 55 −22.18 0.45 0.32 0.20 0.24 0.00 56 −22.16 0.52 0.24 0.000.23 1.00 57 −22.06 0.82 0.27 0.24 0.27 0.79 58 −22.05 0.59 0.30 0.000.22 1.00 59 −22.01 0.36 0.29 0.00 0.00 0.96 60 −21.91 0.62 0.33 0.000.21 1.00 61 −21.88 0.46 0.33 0.21 0.18 0.73 62 −21.83 0.49 0.30 0.240.22 0.99 63 −21.80 0.39 0.29 0.00 0.30 0.51 64 −21.75 0.44 0.28 0.000.24 0.22 65 −21.74 0.58 0.31 0.00 0.26 0.00 66 −21.65 0.52 0.33 0.250.26 0.82 67 −21.59 0.58 0.24 0.00 0.27 0.71 68 −21.58 0.40 0.30 0.260.25 0.85 69 −21.57 0.64 0.25 0.22 0.28 0.96 70 −21.54 0.46 0.31 0.220.00 0.28 71 −21.50 0.73 0.25 0.28 1.00 0.28 72 −21.47 0.34 0.32 0.220.00 0.86 73 −21.43 0.46 0.29 0.22 0.25 0.38 74 −21.41 0.62 0.31 0.000.27 0.85 75 −21.41 0.58 0.28 0.00 0.00 0.29

Example 2: Identification of Protein Fragments Containing Ratios ofEssential Amino Acids, Branch Chain Amino Acids, and Leucine Greaterthan or Equal to Whey

Fragments from the set of 8,415 proteins described in Example 1 wereevaluated for amino acid content, percentage of essential amino acids(“EAA”), the percentage of branched chain amino acids (“BCAA”), thepercentage of leucine (“L”), and whether the protein contained all ofthe essential amino acids were calculated for each protein. In addition,the protein fragments were screened against a database of knownallergens to determine whether any had greater than 50% global homologyto a known allergen. A total of 414 protein fragments were identifiedthat contain greater than or equal to 51% EAA, greater than or equal to25% BCAA, and greater than or equal to 13% Leu, and have less than 50%global homology to known allergens (SEQ ID NOS: 1001 to 1414). For theset of proteins the solvation score at pH 7 (“SolvScore”), aggregationscore at pH 7 (“AggScore”), allergenicity (i.e., percent local homologyto known allergens, as described herein), toxicity (i.e., percenthomology to known toxins, as described herein), anti-nutricity (i.e.,percent homology to known protease inhibitors, as described herein), andhuman homology (i.e., percent homology to known human proteins, asdescribed herein) were calculated, and the total number of Cys residues(“C”) were determined. The characteristics of 75 representative proteinsthus identified are presented in Tables 4A and 4B.

TABLE 4A Seq ID No UniProt FragEnds EAA BCAA L C 1001 P21641  1:126 0.520.30 0.19 1 1002 Q41782 151:276 0.52 0.25 0.14 4 1003 O24174 176:2510.53 0.29 0.15 3 1004 Q41784 201:276 0.55 0.26 0.17 3 1005 P09315276:351 0.51 0.35 0.14 1 1006 Q43695 201:276 0.53 0.26 0.17 3 1007P18025 201:276 0.54 0.26 0.17 3 1008 P18026 201:276 0.54 0.26 0.17 31009 Q43697 201:276 0.54 0.26 0.17 3 1010 Q41783 201:276 0.54 0.26 0.173 1011 Q41785 201:276 0.54 0.26 0.17 3 1012 O24562 126:276 0.53 0.280.14 1 1013 Q6ZHS4 126:301 0.52 0.27 0.13 1 1014 Q6ERW7 226:301 0.550.29 0.13 1 1015 Q01595  51:126 0.52 0.36 0.22 7 1016 Q6ERW9 226:3010.54 0.29 0.13 1 1017 Q0J6T3 151:276 0.55 0.28 0.13 1 1018 P93203201:276 0.55 0.25 0.18 0 1019 Q7XWU3 151:276 0.52 0.27 0.14 1 1020Q41874 126:251 0.51 0.29 0.16 2 1021 Q8H859 151:276 0.54 0.25 0.14 11022 P0CD58 301:476 0.53 0.32 0.18 3 1023 P0CD59 301:476 0.53 0.32 0.183 1024 O04161 251:376 0.58 0.26 0.13 5 1025 Q2R114 151:276 0.54 0.260.13 1 1026 Q5Z987 1051:1276 0.54 0.30 0.14 3 1027 Q41808 176:276 0.510.28 0.17 2 1028 Q41807 176:276 0.51 0.29 0.17 2 1029 P54773 101:2760.55 0.29 0.15 0 1030 Q5Z987 1151:1251 0.60 0.34 0.14 2 1031 Q5Z9871151:1351 0.54 0.33 0.16 4 1032 P31542 326:401 0.53 0.30 0.14 0 1033P31541 126:201 0.51 0.32 0.15 0 1034 P04706  1:76 0.51 0.31 0.17 2 1035O64411  1:76 0.53 0.32 0.18 0 1036 Q0JI49 351:426 0.60 0.26 0.17 0 1037Q9XGC9 476:551 0.57 0.26 0.13 0 1038 P93648 326:401 0.51 0.25 0.17 11039 Q6H7U5 376:451 0.56 0.26 0.14 0 1040 Q43143  76:176 0.52 0.26 0.142 1041 Q9LGV5  51:126 0.52 0.27 0.14 0 1042 P0C8M8 126:201 0.51 0.260.16 4 1043 Q05761  51:126 0.56 0.32 0.16 0 1044 Q84NP7 326:426 0.540.26 0.16 1 1045 Q75HC2 276:351 0.51 0.27 0.14 2 1046 Q6QNU4 126:2010.52 0.27 0.14 2 1047 Q5JMF2  51:126 0.55 0.26 0.14 4 1048 Q7XU38276:351 0.54 0.25 0.16 1 1049 Q8GUQ5 1076:1151 0.53 0.27 0.16 2 1050P51059  76:176 0.52 0.27 0.14 0 1051 P0C8M8  26:176 0.54 0.27 0.14 61052 P49037  26:101 0.51 0.29 0.15 0 1053 O49187 101:176 0.56 0.30 0.180 1054 Q0J4I1  26:176 0.52 0.25 0.14 2 1055 Q6ZAG3  51:176 0.53 0.250.13 1 1056 P29620  51:126 0.54 0.27 0.14 0 1057 P33544 201:276 0.540.26 0.15 1 1058 O24573  1:101 0.55 0.28 0.14 2 1059 Q3HRN8 126:201 0.550.26 0.15 0 1060 Q3HRP5  1:101 0.53 0.27 0.15 3 1061 P93647 801:876 0.530.32 0.16 0 1062 Q9ZS62  951:1026 0.53 0.35 0.16 0 1063 Q6Z4U2 276:3510.53 0.29 0.13 1 1064 P29185 201:301 0.57 0.31 0.13 1 1065 Q75LU8  1:760.51 0.31 0.19 4 1066 Q43298 201:301 0.55 0.31 0.13 1 1067 P49971 26:101 0.52 0.27 0.13 2 1068 P46641  1:76 0.64 0.26 0.16 1 1069 Q2QMX9701:776 0.52 0.25 0.14 0 1070 Q43270 226:301 0.53 0.25 0.13 0 1071P93841  76:176 0.52 0.26 0.15 1 1072 P10581 151:226 0.52 0.29 0.14 11073 P93647 151:251 0.51 0.26 0.14 0 1074 Q2QQS5 126:201 0.52 0.30 0.130 1075 Q0JNK5 301:426 0.52 0.34 0.15 1

TABLE 4B Aller- Tox- Anti- Human Seq ID No SolvScore AggScore genicityicity nutricity Homology 1001 −7.76 1.07 0.63 0.00 0.00 0.28 1002 −16.110.62 0.49 0.24 0.26 0.87 1003 −11.65 0.84 0.48 0.00 0.29 0.54 1004−13.93 0.66 0.46 0.00 0.27 0.87 1005 −15.37 0.78 0.45 0.32 0.00 0.471006 −14.07 0.63 0.45 0.00 0.27 0.86 1007 −14.01 0.63 0.45 0.00 0.270.87 1008 −14.01 0.63 0.45 0.00 0.27 0.87 1009 −14.01 0.63 0.45 0.000.27 0.87 1010 −14.01 0.63 0.45 0.00 0.27 0.87 1011 −14.01 0.63 0.450.00 0.27 0.87 1012 −14.02 0.70 0.44 0.24 0.27 0.29 1013 −13.33 0.730.42 0.00 0.26 0.27 1014 −10.77 0.79 0.42 0.00 0.30 0.34 1015 −14.670.92 0.40 0.31 0.29 0.28 1016 −13.59 0.79 0.39 0.00 0.29 0.35 1017−13.51 0.76 0.39 0.00 0.00 0.28 1018 −35.15 0.19 0.38 0.00 0.00 0.351019 −16.25 0.65 0.38 0.22 0.00 0.27 1020 −13.96 0.54 0.38 0.00 0.000.83 1021 −15.75 0.75 0.37 0.22 0.22 0.27 1022 −9.84 1.10 0.36 0.00 0.250.26 1023 −9.84 1.10 0.36 0.00 0.25 0.26 1024 −9.79 0.97 0.36 0.27 0.240.29 1025 −14.86 0.77 0.36 0.21 0.00 0.27 1026 −20.01 0.60 0.36 0.000.00 0.20 1027 −11.76 0.58 0.36 0.00 0.00 0.85 1028 −11.75 0.58 0.360.00 0.00 0.85 1029 −14.00 0.90 0.36 0.00 0.25 0.26 1030 −19.70 0.840.35 0.00 0.21 0.00 1031 −19.59 0.71 0.35 0.00 0.17 0.24 1032 −24.740.63 0.28 0.67 0.29 0.00 1033 −20.05 0.57 0.24 0.40 0.28 0.30 1034 −4.010.67 0.31 0.40 0.38 0.43 1035 −10.91 1.01 0.29 0.39 0.34 0.42 1036−30.64 0.41 0.27 0.00 0.30 0.00 1037 −27.88 0.25 0.23 0.00 0.00 0.311038 −27.52 0.30 0.27 0.00 0.27 0.64 1039 −27.42 0.47 0.29 0.00 0.000.00 1040 −27.42 0.29 0.32 0.00 0.27 0.00 1041 −26.41 0.31 0.26 0.000.29 0.53 1042 −26.11 0.38 0.28 0.22 0.28 0.66 1043 −25.86 0.56 0.250.00 0.26 0.79 1044 −25.75 0.31 0.32 0.00 0.28 0.58 1045 −25.74 0.510.27 0.29 0.30 0.31 1046 −25.62 0.48 0.26 0.30 0.21 0.66 1047 −25.190.41 0.27 0.32 0.29 0.26 1048 −25.11 0.72 0.28 0.00 0.28 0.36 1049−24.99 0.51 0.21 0.27 0.27 0.34 1050 −24.92 0.42 0.28 0.00 0.00 0.001051 −24.77 0.34 0.30 0.26 0.24 0.50 1052 −24.70 0.55 0.26 0.00 0.220.32 1053 −24.58 0.52 0.25 0.00 0.00 0.00 1054 −24.51 0.39 0.33 0.000.00 0.52 1055 −24.44 0.27 0.31 0.26 0.00 0.46 1056 −24.32 0.51 0.270.00 0.00 0.56 1057 −24.32 0.57 0.25 0.00 0.31 0.00 1058 −24.14 0.550.31 0.00 0.28 0.48 1059 −24.14 0.48 0.29 0.00 0.30 0.41 1060 −23.960.56 0.34 0.00 0.26 0.30 1061 −23.86 0.55 0.26 0.00 0.26 0.58 1062−23.81 0.67 0.23 0.00 0.24 0.00 1063 −23.35 0.63 0.23 0.26 0.26 0.281064 −23.30 0.56 0.28 0.00 0.00 0.56 1065 −23.28 0.63 0.33 0.26 0.300.27 1066 −23.25 0.56 0.29 0.00 0.27 0.55 1067 −23.24 0.32 0.28 0.290.28 0.50 1068 −23.20 0.71 0.29 0.33 0.25 0.27 1069 −22.91 0.44 0.270.00 0.30 0.53 1070 −22.86 0.47 0.28 0.31 0.25 0.34 1071 −22.79 0.510.29 0.25 0.25 0.25 1072 −22.76 0.62 0.25 0.00 0.00 0.00 1073 −22.760.47 0.30 0.00 0.00 0.31 1074 −22.72 0.56 0.27 0.00 0.21 0.31 1075−22.70 0.75 0.30 0.00 0.00 0.51

Example 3: Identification of Protein Fragments Containing Ratios ofEssential Amino Acids and Branch Chain Amino Acids Greater than or Equalto Whey and Containing all Essential Amino Acids

Fragments from the set of 8,415 proteins described in Example 1 wereevaluated for amino acid content, percentage of essential amino acids(“EAA”), the percentage of branched chain amino acids (“BCAA”), thepercentage of leucine (“L”), and whether the protein contained all ofthe essential amino acids were calculated for each protein. In addition,the protein fragments were screened against a database of knownallergens to determine whether any had greater than 50% global homologyto a known allergen. A total of 485 proteins were identified thatcontain greater than or equal to 51% EAA and greater than or equal to25% BCAA, and that contain all essential amino acids, and have less than50% global homology to known allergens (SEQ ID NOS: 1415 to 1899). Forthe set of protein fragments the solvation score at pH 7 (“SolvScore”),aggregation score at pH 7 (“AggScore”), allergenicity (i.e., percentlocal homology to known allergens, as described herein), toxicity (i.e.,percent homology to known toxins, as described herein), anti-nutricity(i.e., percent homology to known protease inhibitors, as describedherein), and human homology (i.e., percent homology to known humanproteins, as described herein) were calculated, and the total number ofCys residues (“C”) were determined. The characteristics of 75representative proteins thus identified are presented in Table 5A and5B.

TABLE 5A Seq ID No UniProt FragEnds EAA BCAA L C 1415 Q96576 151:3260.52 0.27 0.10 2 1416 P15722 176:276 0.53 0.28 0.04 1 1417 Q8GUQ5 876:1001 0.51 0.25 0.12 1 1418 P17801 476:751 0.52 0.26 0.12 3 1419Q6ERW7 201:351 0.53 0.26 0.10 1 1420 Q7XWU3  51:276 0.51 0.25 0.09 61421 Q6ER94  76:226 0.52 0.26 0.10 1 1422 Q43272 176:376 0.52 0.26 0.106 1423 Q43272  76:326 0.52 0.28 0.11 7 1424 Q6ERW9 226:351 0.55 0.260.11 1 1425 Q10717 251:326 0.51 0.30 0.10 1 1426 Q6ERW9 126:276 0.510.25 0.06 5 1427 Q6ERW5  26:101 0.52 0.25 0.03 4 1428 P16243 226:4760.51 0.27 0.12 4 1429 Q43272  76:226 0.51 0.28 0.12 4 1430 O04161151:376 0.55 0.25 0.11 5 1431 Q9ATN1 101:251 0.56 0.29 0.11 2 1432Q9ATN1  1:251 0.51 0.25 0.10 5 1433 P29185  26:301 0.52 0.25 0.08 3 1434Q08451 126:276 0.53 0.27 0.08 2 1435 Q96575  1:276 0.52 0.26 0.12 4 1436P09607  1:276 0.51 0.26 0.12 5 1437 Q6ETN3 401:501 0.52 0.25 0.06 0 1438Q5BQN5 151:226 0.60 0.26 0.11 1 1439 Q2MI42  976:1176 0.64 0.25 0.10 01440 P28523  26:176 0.51 0.26 0.09 3 1441 Q8H6B1 126:226 0.57 0.26 0.101 1442 P54767 176:251 0.54 0.26 0.11 1 1443 Q67W82 401:526 0.51 0.260.06 0 1444 P0C8M8  1:226 0.53 0.25 0.11 7 1445 Q9FYT6 551:651 0.53 0.250.09 0 1446 P0C8M8 151:226 0.52 0.26 0.12 2 1447 Q10S72 426:551 0.520.26 0.10 1 1448 Q42982 426:551 0.53 0.26 0.06 0 1449 Q2MI46  26:1010.51 0.25 0.09 4 1450 B4G0F3 276:401 0.52 0.26 0.07 1 1451 P93648 76:251 0.52 0.26 0.12 1 1452 C1K5M3 551:751 0.55 0.25 0.12 5 1453Q41741 151:276 0.53 0.27 0.12 1 1454 O22424 151:251 0.60 0.25 0.05 01455 Q9XFH3  51:201 0.54 0.26 0.10 3 1456 Q7XTS4 201:451 0.51 0.25 0.113 1457 Q9FUZ0 176:276 0.52 0.25 0.12 2 1458 P05118  1:76 0.59 0.26 0.101 1459 Q8S7E1  51:126 0.52 0.27 0.12 1 1460 C1K5M2 101:201 0.55 0.250.10 3 1461 Q6Z4U2 151:351 0.52 0.26 0.12 4 1462 Q8H6B1  26:176 0.560.25 0.11 0 1463 Q0J7J7 176:351 0.51 0.27 0.11 5 1464 P10581 651:7510.51 0.27 0.12 1 1465 Q2RAS0  26:251 0.51 0.25 0.09 2 1466 O24594351:426 0.53 0.29 0.08 2 1467 Q0J7J7 276:351 0.54 0.31 0.12 3 1468Q6XZ79 176:276 0.51 0.25 0.10 1 1469 P19862 651:801 0.53 0.26 0.11 31470 P28554 276:426 0.53 0.26 0.11 2 1471 C1K5M3 451:651 0.52 0.25 0.124 1472 P31927 451:551 0.51 0.26 0.12 1 1473 Q65X71 451:526 0.61 0.310.12 0 1474 Q00497  76:226 0.51 0.25 0.11 4 1475 Q7XKC0  76:176 0.510.26 0.06 3 1476 P10582 276:451 0.55 0.27 0.12 3 1477 P29535 176:4010.51 0.26 0.11 8 1478 Q43298  26:301 0.51 0.25 0.08 3 1479 Q7XRA1176:251 0.61 0.29 0.12 0 1480 Q852N2  826:1001 0.52 0.27 0.12 6 1481Q65X71 451:726 0.52 0.26 0.09 5 1482 Q852N2 826:901 0.51 0.25 0.10 21483 Q65X71  51:326 0.52 0.27 0.10 3 1484 Q8H6B1 801:901 0.57 0.29 0.121 1485 P54767 176:451 0.52 0.26 0.10 2 1486 P61242 1626:1876 0.51 0.270.12 4 1487 Q65X71 451:626 0.54 0.26 0.09 3 1488 P04712 326:426 0.520.27 0.11 1 1489 Q75LR2 351:426 0.55 0.28 0.12 0

TABLE 5B Aller- Tox- Anti- Human Seq ID No SolvScore AggScore genicityicity nutricity Homology 1415 −16.93 0.58 0.54 0.53 0.24 0.00 1416−19.58 0.41 0.53 0.00 0.21 0.00 1417 −21.91 0.46 0.47 0.27 0.27 0.371418 −19.73 0.54 0.46 0.00 0.00 0.34 1419 −11.15 0.74 0.43 0.00 0.250.29 1420 −16.57 0.61 0.42 0.22 0.00 0.27 1421 −20.09 0.54 0.41 0.230.00 0.63 1422 −16.36 0.68 0.41 0.00 0.24 0.34 1423 −16.55 0.67 0.400.00 0.21 0.32 1424 −11.31 0.79 0.40 0.24 0.00 0.27 1425 −17.05 0.640.39 0.28 0.35 0.53 1426 −11.49 0.73 0.38 0.00 0.26 0.35 1427 −17.250.63 0.38 0.25 0.28 0.41 1428 −19.07 0.59 0.38 0.21 0.00 0.60 1429−16.46 0.67 0.36 0.25 0.23 0.33 1430 −11.00 0.89 0.36 0.00 0.00 0.261431 −6.06 1.08 0.36 0.27 0.27 0.36 1432 −8.76 0.87 0.36 0.23 0.23 0.281433 −21.28 0.49 0.36 0.00 0.00 0.60 1434 −10.55 0.95 0.35 0.25 0.240.44 1435 −17.94 0.61 0.34 0.37 0.00 0.00 1436 −18.54 0.63 0.33 0.360.19 0.00 1437 −28.54 0.46 0.30 0.00 0.23 0.38 1438 −25.54 0.40 0.270.00 0.00 0.26 1439 −25.12 0.37 0.33 0.24 0.00 0.19 1440 −25.09 0.340.31 0.00 0.21 0.80 1441 −24.93 0.38 0.32 0.00 0.25 0.27 1442 −24.550.45 0.22 0.00 0.00 0.32 1443 −24.41 0.49 0.29 0.24 0.00 0.33 1444−24.26 0.36 0.31 0.00 0.00 0.49 1445 −23.95 0.53 0.28 0.00 0.00 0.001446 −23.76 0.45 0.30 0.00 0.26 0.70 1447 −23.69 0.45 0.30 0.24 0.270.39 1448 −23.58 0.48 0.31 0.00 0.24 0.36 1449 −23.51 0.56 0.27 0.250.27 0.36 1450 −23.40 0.48 0.31 0.25 0.00 0.43 1451 −23.21 0.41 0.300.00 0.22 0.27 1452 −23.11 0.50 0.30 0.00 0.23 0.00 1453 −22.98 0.530.30 0.29 0.26 0.71 1454 −22.62 0.40 0.27 0.00 0.00 0.70 1455 −22.540.48 0.31 0.24 0.24 0.42 1456 −22.52 0.37 0.33 0.24 0.00 0.30 1457−22.50 0.44 0.30 0.00 0.00 0.44 1458 −22.49 0.61 0.27 0.24 1.00 0.261459 −22.48 0.47 0.23 0.00 0.27 0.24 1460 −22.30 0.61 0.30 0.23 0.190.36 1461 −22.29 0.45 0.30 0.21 0.24 0.00 1462 −22.25 0.48 0.31 0.000.21 0.29 1463 −22.20 0.44 0.30 0.00 0.22 0.00 1464 −22.05 0.53 0.280.00 0.00 0.00 1465 −22.05 0.69 0.30 0.00 0.00 0.29 1466 −22.02 0.450.29 0.00 0.32 0.54 1467 −22.00 0.48 0.27 0.00 0.30 0.32 1468 −21.970.50 0.29 0.00 0.00 0.31 1469 −21.83 0.54 0.30 0.23 0.00 0.00 1470−21.80 0.58 0.31 0.25 0.00 0.23 1471 −21.78 0.56 0.30 0.21 0.23 0.001472 −21.60 0.54 0.30 0.00 0.00 0.00 1473 −21.53 0.69 0.24 0.00 0.260.33 1474 −21.44 0.48 0.33 0.21 0.00 0.27 1475 −21.39 0.65 0.28 0.260.00 0.59 1476 −21.27 0.57 0.30 0.00 0.24 0.00 1477 −21.26 0.50 0.330.00 0.23 0.30 1478 −21.26 0.49 0.32 0.00 0.00 0.60 1479 −21.13 0.550.28 0.00 0.00 0.28 1480 −21.08 0.61 0.33 0.17 0.24 0.27 1481 −21.000.50 0.31 0.19 0.22 0.39 1482 −20.97 0.43 0.24 0.17 0.26 0.00 1483−20.94 0.68 0.33 0.00 0.23 0.34 1484 −20.89 0.61 0.29 0.00 0.26 0.511485 −20.84 0.48 0.31 0.00 0.00 0.23 1486 −20.74 0.51 0.30 0.00 0.220.22 1487 −20.65 0.51 0.31 0.28 0.27 0.34 1488 −20.58 0.44 0.32 0.000.00 0.00 1489 −20.54 0.44 0.28 0.00 0.00 0.21

Example 4: Identification of Protein Fragments Containing Ratios ofEssential Amino Acids and Leucine Greater than or Equal to Whey andContaining all Essential Amino Acids

Fragments from the set of 8,415 proteins described in Example 1 wereevaluated for amino acid content, percentage of essential amino acids(“EAA”), the percentage of branched chain amino acids (“BCAA”), thepercentage of leucine (“L”), and whether the protein contained all ofthe essential amino acids were calculated for each protein. In addition,the protein fragments were screened against a database of knownallergens to determine whether any had greater than 50% global homologyto a known allergen. A total of 203 protein fragments were identifiedthat contain greater than or equal to 51% EAA and greater than or equalto 13% Leu, and that contain all essential amino acids, and have lessthan 50% global homology to known allergens (SEQ ID NOS: 1900 to 2102).For the set of protein fragments the solvation score at pH 7(“SolvScore”), aggregation score at pH 7 (“AggScore”), allergenicity(i.e., percent local homology to known allergens, as described herein),toxicity (i.e., percent homology to known toxins, as described herein),anti-nutricity (i.e., percent homology to known protease inhibitors, asdescribed herein), and human homology (i.e., percent homology to knownhuman proteins, as described herein) were calculated, and the totalnumber of Cys residues (“C”) were determined. The characteristics of 75representative protein fragments thus identified are presented in Table6A and 6B.

TABLE 6A Seq ID No UniProt FragEnds EAA BCAA L C 1900 Q41784 201:3010.51 0.22 0.15 4 1901 P18025 201:301 0.51 0.22 0.15 4 1902 Q43697201:301 0.51 0.22 0.15 4 1903 Q41783 201:301 0.51 0.22 0.15 4 1904Q41785 201:301 0.51 0.22 0.15 4 1905 P18026 201:301 0.51 0.22 0.15 41906 P10708  76:176 0.51 0.24 0.13 1 1907 P27525  51:151 0.53 0.22 0.140 1908 Q3HRN9  51:201 0.52 0.23 0.14 1 1909 Q9ZSV1 726:801 0.51 0.230.14 1 1910 Q5BQN5  51:176 0.52 0.20 0.13 1 1911 P56669  1:76 0.53 0.180.14 1 1912 Q75LU8  1:201 0.52 0.24 0.13 4 1913 Q8S0F0 601:851 0.51 0.230.14 3 1914 Q9ZSV1 626:801 0.51 0.23 0.13 1 1915 Q0JI49 301:451 0.530.23 0.14 0 1916 P93703 201:401 0.51 0.24 0.14 1 1917 Q2MI42 426:6010.52 0.23 0.13 1 1918 Q8S0F0 501:751 0.52 0.23 0.13 4 1919 Q2MI421601:1701 0.51 0.23 0.14 1 1920 Q7XTS3 401:476 0.57 0.21 0.13 2 1921P49037  1:201 0.51 0.24 0.14 1 1922 Q9FQ11  1:76 0.53 0.24 0.16 1 1923Q7X996 201:376 0.55 0.22 0.13 1 1924 P27061 126:201 0.53 0.24 0.14 01925 P53682 326:401 0.51 0.24 0.13 1 1926 Q84ZL0 1201:1476 0.51 0.240.16 4 1927 Q8S0F0 701:851 0.51 0.24 0.14 1 1928 Q6ZAG3  51:201 0.540.24 0.13 1 1929 Q69Q02 276:451 0.52 0.22 0.13 1 1930 P10581 276:5260.51 0.24 0.13 2 1931 P49037 201:276 0.52 0.24 0.15 2 1932 P61242776:951 0.51 0.23 0.15 2 1933 Q5Z987 1826:1926 0.51 0.19 0.14 1 1934Q5JMM1 251:326 0.52 0.22 0.14 0 1935 Q84N48 426:526 0.51 0.24 0.14 01936 Q7G6K7  826:1101 0.51 0.23 0.14 4 1937 Q0J4I1  1:201 0.53 0.24 0.142 1938 Q7XWS7 1226:1476 0.51 0.22 0.13 3 1939 Q0JF02  76:276 0.51 0.240.14 4 1940 Q2QN26 201:351 0.51 0.24 0.14 5 1941 P49037 101:276 0.530.22 0.14 2 1942 Q6K5F8 351:626 0.51 0.24 0.14 3 1943 Q10QA2 101:2260.51 0.22 0.13 6 1944 Q9XHR2 201:301 0.52 0.24 0.14 0 1945 Q0DCT8176:351 0.52 0.24 0.15 5 1946 Q6AU53 476:576 0.52 0.24 0.16 0 1947A7LFZ6 276:451 0.52 0.23 0.15 6 1948 Q6ZKB2 451:726 0.52 0.22 0.13 21949 Q8S983 176:251 0.51 0.22 0.14 0 1950 Q3LXA7 526:626 0.53 0.24 0.142 1951 P93703 201:276 0.55 0.24 0.14 1 1952 A3B9A0  76:226 0.51 0.210.13 2 1953 Q9XGD6  51:126 0.52 0.22 0.13 0 1954 P18485 301:376 0.540.20 0.14 2 1955 Q32SG6 876:951 0.52 0.23 0.16 0 1956 Q5VS72 501:5760.54 0.24 0.18 2 1957 Q43503 401:476 0.54 0.21 0.15 1 1958 Q42896101:326 0.52 0.24 0.13 4 1959 Q9MAX6 151:251 0.52 0.24 0.14 3 1960Q9LKX9  1:201 0.52 0.22 0.14 5 1961 P09562 176:301 0.52 0.23 0.15 3 1962Q7XWS7 1226:1376 0.51 0.22 0.13 1 1963 Q7XU38 151:351 0.51 0.24 0.14 41964 Q69LX2  926:1151 0.51 0.24 0.13 5 1965 Q7XD96 601:726 0.51 0.220.14 5 1966 Q7XI73  26:101 0.52 0.17 0.13 5 1967 Q42881 301:376 0.530.20 0.14 3 1968 C1K5M3 176:301 0.53 0.23 0.14 4 1969 Q6L4F8 226:3010.53 0.22 0.15 2 1970 P48022  1:101 0.51 0.23 0.14 0 1971 Q8LHA8  51:2010.51 0.24 0.14 3 1972 Q6ZAG3 151:301 0.52 0.24 0.14 3 1973 P04711751:901 0.53 0.24 0.13 0 1974 Q5N870 576:801 0.53 0.23 0.13 8

TABLE 6B Aller- Tox- Anti- Human Seq ID No SolvScore AggScore genicityicity nutricity Homology 1900 −14.31 0.56 0.48 0.00 0.25 0.88 1901−14.31 0.54 0.46 0.00 0.25 0.88 1902 −14.31 0.54 0.46 0.00 0.25 0.881903 −14.31 0.54 0.46 0.00 0.25 0.88 1904 −14.31 0.54 0.46 0.00 0.250.88 1905 −14.42 0.53 0.46 0.00 0.24 0.87 1906 −16.21 0.63 0.41 0.000.00 0.28 1907 −14.10 0.61 0.39 0.00 0.25 0.00 1908 −24.00 0.41 0.360.22 0.26 0.31 1909 −27.98 0.27 0.25 0.29 0.33 0.49 1910 −27.07 0.170.33 0.23 0.24 0.00 1911 −27.03 0.25 0.22 0.00 0.27 0.41 1912 −25.610.41 0.35 0.23 0.25 0.32 1913 −24.50 0.36 0.33 0.00 0.00 0.30 1914−24.43 0.35 0.35 0.00 0.23 0.39 1915 −24.41 0.37 0.32 0.24 0.23 0.001916 −24.40 0.42 0.32 0.00 0.24 0.30 1917 −24.34 0.23 0.33 0.00 0.230.00 1918 −23.77 0.39 0.32 0.00 0.00 0.34 1919 −23.65 0.44 0.31 0.000.22 0.00 1920 −23.49 0.37 0.28 0.00 0.00 0.33 1921 −23.32 0.40 0.330.22 0.25 0.00 1922 −23.31 0.48 0.25 0.26 0.24 0.00 1923 −23.23 0.390.30 0.00 0.21 0.27 1924 −23.18 0.42 0.24 0.00 0.00 0.00 1925 −23.130.29 0.28 0.25 0.26 0.27 1926 −23.12 0.37 0.34 0.00 0.00 0.32 1927−23.00 0.35 0.34 0.00 0.25 0.29 1928 −22.83 0.31 0.31 0.00 0.22 0.491929 −22.74 0.36 0.29 0.00 0.23 0.00 1930 −22.67 0.39 0.31 0.00 0.000.00 1931 −22.62 0.50 0.26 0.25 0.27 0.26 1932 −22.44 0.44 0.33 0.240.22 0.00 1933 −22.39 0.41 0.31 0.22 0.25 0.34 1934 −22.25 0.36 0.220.00 0.33 0.33 1935 −22.24 0.40 0.30 0.00 0.25 0.00 1936 −22.14 0.350.32 0.00 0.00 0.31 1937 −22.12 0.41 0.34 0.00 0.00 0.48 1938 −21.990.35 0.31 0.00 0.00 0.29 1939 −21.98 0.49 0.33 0.00 0.23 0.00 1940−21.79 0.55 0.32 0.26 0.24 0.36 1941 −21.63 0.40 0.29 0.00 0.21 0.001942 −21.60 0.42 0.33 0.00 0.24 0.65 1943 −21.54 0.62 0.30 0.25 0.240.33 1944 −21.23 0.37 0.28 0.00 0.00 0.42 1945 −21.20 0.48 0.32 0.000.25 0.40 1946 −21.19 0.55 0.30 0.00 0.25 0.00 1947 −21.18 0.43 0.300.00 0.25 0.00 1948 −21.10 0.36 0.32 0.00 0.00 0.31 1949 −20.98 0.410.24 0.00 0.26 0.24 1950 −20.85 0.56 0.30 0.00 0.24 0.37 1951 −20.780.47 0.26 0.00 0.23 0.31 1952 −20.67 0.40 0.30 0.23 0.22 0.00 1953−20.65 0.55 0.26 0.00 0.00 0.37 1954 −20.62 0.51 0.28 0.28 0.26 0.301955 −20.55 0.33 0.26 0.00 0.29 0.00 1956 −20.51 0.45 0.26 0.28 0.000.46 1957 −20.51 0.66 0.26 0.00 0.25 0.26 1958 −20.48 0.48 0.32 0.000.20 0.25 1959 −20.40 0.55 0.32 0.00 0.26 0.56 1960 −20.37 0.48 0.290.24 0.22 0.00 1961 −20.36 0.45 0.29 0.25 0.23 0.00 1962 −20.34 0.330.30 0.23 0.24 0.30 1963 −20.33 0.59 0.30 0.00 0.22 0.25 1964 −20.310.50 0.30 0.21 0.20 0.25 1965 −20.18 0.36 0.29 0.30 0.21 0.32 1966−20.15 0.38 0.28 0.31 0.29 0.27 1967 −20.14 0.50 0.31 0.00 0.27 0.361968 −20.11 0.36 0.27 0.00 0.00 0.00 1969 −19.97 0.40 0.23 0.23 0.280.63 1970 −19.89 0.73 0.30 0.00 0.00 0.29 1971 −19.81 0.55 0.32 0.220.22 0.31 1972 −19.49 0.46 0.33 0.00 0.25 0.51 1973 −19.43 0.48 0.340.26 0.20 0.00 1974 −19.42 0.46 0.32 0.00 0.00 0.26

Example 5: Identification of Proteins Containing Ratios of Branch ChainAmino Acids and Leucine Greater than or Equal to Whey and Containing allEssential Amino Acids

Fragments from the set of 8,415 proteins described in Example 1 wereevaluated for amino acid content, percentage of essential amino acids(“EAA”), the percentage of branched chain amino acids (“BCAA”), thepercentage of leucine (“L”), and whether the protein contained all ofthe essential amino acids were calculated for each protein. In addition,the proteins were screened against a database of known allergens todetermine whether any had greater than 50% global homology to a knownallergen. A total of 415 protein fragments were identified that containgreater than or equal to 25% BCAA and greater than or equal to 13% Leu,and that contain all essential amino acids, and have less than 50%global homology to known allergens (SEQ ID NOS: 2103 to 2518). For theset of protein fragments the solvation score at pH 7 (“SolvScore”),aggregation score at pH 7 (“AggScore”), allergenicity (i.e., percentlocal homology to known allergens, as described herein), toxicity (i.e.,percent homology to known toxins, as described herein), anti-nutricity(i.e., percent homology to known protease inhibitors, as describedherein), and human homology (i.e., percent homology to known humanproteins, as described herein) were calculated, and the total number ofCys residues (“C”) were determined. The characteristics of 75representative protein fragments thus identified are presented in Table7A and 7B.

TABLE 7A Seq ID No UniProt FragEnds EAA BCAA L C 2103 O24174  76:2510.49 0.26 0.14 4 2104 P24396  1:126 0.48 0.27 0.13 2 2105 P17801 501:7760.49 0.26 0.13 3 2106 O24585 501:751 0.47 0.26 0.14 3 2107 O24585626:751 0.46 0.29 0.14 1 2108 P42211  1:101 0.50 0.27 0.20 2 2109 Q75L42 51:226 0.48 0.28 0.13 3 2110 Q9LGV5  1:251 0.48 0.26 0.13 4 2111 Q9LGV5101:226 0.45 0.26 0.14 3 2112 Q10PS6 101:276 0.47 0.26 0.13 5 2113Q5JLQ9  76:226 0.47 0.26 0.15 3 2114 Q2QMI0  26:226 0.43 0.26 0.15 32115 Q0D4B2 201:301 0.47 0.27 0.14 3 2116 Q8GUQ5 351:626 0.49 0.30 0.206 2117 Q8GUQ5 451:626 0.47 0.31 0.22 3 2118 Q8GUQ5 451:726 0.46 0.280.18 4 2119 Q2QY53 126:226 0.46 0.25 0.17 3 2120 Q2RAX3 126:226 0.460.25 0.17 3 2121 Q75GK4  1:226 0.46 0.25 0.16 3 2122 P16243 176:376 0.490.28 0.13 4 2123 Q41807 151:401 0.49 0.26 0.13 3 2124 Q69Q47  51:2260.49 0.27 0.14 3 2125 P46420  1:201 0.48 0.25 0.13 3 2126 Q6Z9F4  51:2260.47 0.27 0.13 3 2127 P16025 1026:1226 0.50 0.26 0.14 3 2128 P16025 926:1151 0.45 0.26 0.13 2 2129 P49174  1:76 0.47 0.29 0.14 0 2130P48186  1:176 0.49 0.25 0.13 1 2131 P93648 276:401 0.50 0.26 0.16 2 2132P49037 551:626 0.49 0.26 0.15 1 2133 Q2MIA9 201:276 0.48 0.28 0.15 12134 P16024 201:276 0.48 0.28 0.15 1 2135 P16024 201:376 0.44 0.26 0.132 2136 P49037  1:101 0.49 0.27 0.14 1 2137 Q84N48 376:451 0.49 0.27 0.132 2138 Q94IW5 251:351 0.45 0.27 0.14 1 2139 Q75LD5 576:726 0.44 0.250.13 1 2140 Q84ZX8  1:76 0.47 0.25 0.17 0 2141 Q336R3  26:101 0.43 0.260.20 3 2142 P93231 576:851 0.48 0.26 0.14 6 2143 P93648 276:551 0.480.25 0.13 4 2144 P16024 201:476 0.47 0.28 0.13 3 2145 Q2NM15  26:2510.47 0.26 0.16 3 2146 P93231 576:751 0.50 0.25 0.14 3 2147 C7E5V8 26:251 0.47 0.26 0.16 3 2148 C7E5V7  26:251 0.48 0.27 0.17 3 2149P30792 126:226 0.49 0.28 0.15 0 2150 Q5VS72 151:276 0.46 0.25 0.14 12151 Q9XGC9 426:501 0.45 0.27 0.16 0 2152 P61242 1626:1776 0.47 0.280.14 3 2153 Q9XGD6 126:251 0.46 0.27 0.14 2 2154 B6SJQ0  1:176 0.40 0.260.16 4 2155 P93207  1:126 0.42 0.25 0.14 2 2156 Q653U3 801:876 0.44 0.260.14 1 2157 P51059 351:576 0.44 0.25 0.15 4 2158 P07920  76:226 0.480.25 0.15 2 2159 P16024 301:451 0.48 0.28 0.13 2 2160 P29390 176:2510.50 0.25 0.14 1 2161 Q5VQ78 576:676 0.47 0.25 0.14 0 2162 P16025 926:1026 0.46 0.27 0.13 1 2163 P51059 476:576 0.46 0.25 0.15 1 2164P17847 326:451 0.38 0.25 0.16 1 2165 P05116  76:226 0.47 0.25 0.15 22166 P24157  76:226 0.47 0.25 0.15 2 2167 Q9XGC9 301:401 0.48 0.25 0.151 2168 P04711 351:551 0.45 0.25 0.13 3 2169 Q84ZW8 376:551 0.50 0.260.14 5 2170 Q6H6V4 801:876 0.45 0.26 0.13 1 2171 Q9XGD6  26:251 0.480.25 0.14 2 2172 Q41342 301:401 0.49 0.27 0.15 0 2173 Q42883 451:6260.45 0.25 0.14 6 2174 Q2MI98  26:201 0.45 0.25 0.13 1 2175 Q7XTS4251:426 0.49 0.26 0.13 3 2176 Q5Z987 1276:1451 0.47 0.25 0.14 8 2177Q6AVM3 276:351 0.47 0.26 0.16 2

TABLE 7B Aller- Tox- Anti- Human Seq ID No SolvScore AggScore genicityicity nutricity Homology 2103 −17.26 0.66 0.55 0.00 0.27 0.46 2104−14.60 0.68 0.49 0.25 0.26 0.23 2105 −21.22 0.41 0.46 0.00 0.00 0.362106 −19.98 0.48 0.44 0.20 0.23 0.35 2107 −18.81 0.59 0.44 0.00 0.000.40 2108 −15.83 0.61 0.43 0.29 0.27 0.40 2109 −20.27 0.45 0.42 0.000.25 0.55 2110 −21.01 0.39 0.42 0.00 0.00 0.51 2111 −19.90 0.44 0.420.25 0.00 0.59 2112 −13.77 0.63 0.41 0.00 0.19 0.28 2113 −18.33 0.590.40 0.27 0.23 0.53 2114 −19.91 0.56 0.40 0.25 0.25 0.44 2115 −17.670.47 0.39 0.00 0.24 0.59 2116 −12.97 0.46 0.39 0.00 0.24 0.32 2117−12.09 0.48 0.39 0.00 0.24 0.35 2118 −13.41 0.44 0.39 0.00 0.22 0.312119 −17.69 0.50 0.38 0.00 0.00 0.55 2120 −17.69 0.50 0.38 0.00 0.000.55 2121 −18.70 0.55 0.38 0.21 0.00 0.42 2122 −18.11 0.64 0.38 0.000.24 0.61 2123 −16.10 0.45 0.38 0.00 0.00 0.78 2124 −19.89 0.47 0.370.24 0.23 0.48 2125 −18.94 0.60 0.37 0.24 0.25 0.28 2126 −21.69 0.540.37 0.19 0.25 0.52 2127 −17.96 0.48 0.36 0.24 0.00 0.00 2128 −19.800.48 0.36 0.20 0.00 0.00 2129 −10.98 0.80 0.36 0.31 0.28 0.34 2130−20.67 0.47 0.35 0.00 0.23 0.00 2131 −29.02 0.26 0.33 0.26 0.24 0.602132 −28.96 0.53 0.26 0.00 0.27 0.26 2133 −28.54 0.45 0.28 0.23 0.260.30 2134 −28.35 0.49 0.25 0.27 0.00 0.23 2135 −27.16 0.36 0.31 0.000.24 0.28 2136 −25.78 0.45 0.33 0.00 0.25 0.30 2137 −25.22 0.53 0.270.19 0.00 0.33 2138 −24.85 0.53 0.30 0.00 0.26 0.29 2139 −24.69 0.520.33 0.20 0.00 0.00 2140 −24.65 0.46 0.24 0.00 0.30 0.27 2141 −24.350.46 0.24 0.29 0.27 0.27 2142 −24.29 0.47 0.30 0.00 0.00 0.40 2143−24.22 0.34 0.33 0.22 0.00 0.67 2144 −23.92 0.43 0.31 0.21 0.25 0.272145 −23.69 0.40 0.32 0.00 0.00 0.00 2146 −23.69 0.50 0.30 0.00 0.200.43 2147 −23.61 0.41 0.32 0.00 0.00 0.23 2148 −23.59 0.42 0.33 0.000.00 0.00 2149 −23.57 0.39 0.31 0.00 0.27 0.00 2150 −23.48 0.33 0.300.00 0.24 0.34 2151 −23.44 0.29 0.30 0.00 0.25 0.26 2152 −23.42 0.510.30 0.00 0.23 0.27 2153 −23.41 0.52 0.29 0.27 0.00 0.40 2154 −23.340.47 0.32 0.00 0.26 0.00 2155 −23.10 0.47 0.34 0.20 0.22 0.54 2156−23.09 0.45 0.28 0.25 0.28 0.00 2157 −23.07 0.47 0.32 0.00 0.21 0.002158 −23.01 0.33 0.31 0.00 0.25 0.24 2159 −22.95 0.47 0.31 0.24 0.210.32 2160 −22.81 0.26 0.26 0.00 0.00 0.42 2161 −22.75 0.46 0.33 0.000.21 0.53 2162 −22.74 0.44 0.27 0.27 0.28 0.00 2163 −22.72 0.54 0.300.00 0.28 0.24 2164 −22.69 0.36 0.30 0.24 0.00 0.32 2165 −22.64 0.340.31 0.25 0.00 0.00 2166 −22.64 0.34 0.31 0.25 0.00 0.00 2167 −22.520.32 0.29 0.00 0.00 0.47 2168 −22.29 0.48 0.31 0.00 0.25 0.00 2169−22.23 0.51 0.32 0.19 0.22 0.00 2170 −22.16 0.48 0.29 0.25 0.27 0.222171 −22.13 0.49 0.32 0.00 0.00 0.38 2172 −22.05 0.63 0.34 0.00 0.000.00 2173 −21.94 0.45 0.34 0.00 0.26 0.37 2174 −21.89 0.34 0.29 0.000.21 0.00 2175 −21.84 0.41 0.33 0.00 0.25 0.28 2176 −21.74 0.50 0.320.00 0.25 0.30 2177 −21.74 0.50 0.32 0.30 0.28 0.38

Example 6: Protein Expression

Genes encoding nutritive proteins disclosed herein were codon optimizedfor expression in Escherichia coli and synthesized by eitherLifeTechnologies/GeneArt or DNA 2.0. Genes were designed to contain oneof two amino-terminal tags to facilitate purification:

(SEQ ID NO: 2615) MGSHHHHHHHH (SEQ ID NO: 2614) MGSSHHHHHHSSGLVPRGSH

These gene constructs were inserted into the pET15b plasmid vector(Novagen) using NcoI-BamHI restriction sites (in case of the first tag)or using the NdeI-BamHI restriction sites (in the case of the secondtag). All restriction enzymes were purchased from New England Biolabs.Plasmids were transformed into Escherichia coli T7 Express (New EnglandBiolabs) and selected on lysogeny broth (LB) plates containing 100 mg/lcarbenicillin. A single colony was picked, grown to OD_(600 nm)≈0.6 inLB with 100 mg/l carbencillin, and stored as a glycerol stock (in LBwith 10% glycerol (v/v)) at −80° C., to serve as a master cell stock.

2 ml LB with 100 mg/l carbenicillin (in a 14 mm×100 mm culture tube) wasinoculated with a stab from the glycerol stock and grown overnight at37° C. and 250 rpm. The next day, 2 ml LB with 100 mg/l carbenicillin(in a 14 mm×100 mm culture tube) was inoculated with the overnightculture to OD_(600 nm)=0.05 and grown at 30° C. or 37° C. and 250 rpm.At OD_(600 nm)≈0.8, heterologous gene-expression was initiated with 1 mMisopropyl P-D-1-thiogalactopyranoside (IPTG) and grown for another 2 hr(when grown at 37° C.) or 4 hr (when grown at 30° C.) until harvest.Upon harvesting, OD_(600 nm) was measured, a 1 ml aliquot wascentrifuged, and the supernatant was decanted. Cells were re-suspendedto OD_(600 nm)=1.50 for SDS-PAGE analysis to evaluate expression level.10 μl of resuspended culture was loaded onto either: 1) a Novex® NuPAGE®12% Bis-Tris gel (Life Technologies), or 2) a Novex®16% Tricine gel(Life Technologies), and run using standard manufacturer's protocols.Gels were stained using SimplyBlue™ SafeStain (Life Technologies) usingthe standard manufacturer's protocol and imaged using the MolecularImager® Gel Doc™ XR+ System (Bio-Rad). Over-expressed heterologousprotein was identified by comparison against a molecular weight markerand control cultures.

Using this method, recombinant expression of the proteins listed inTables 8A and 8B was observed.

TABLE 8A Seq ID No UniProt FragEnds EAA BCAA L C 2519 P15989 388:4870.52 0.36 0.15 0 2520 P15989 341:442 0.48 0.36 0.16 0 2521 — — 0.50 0.410.16 1 2522 P10587 1287:1386 0.49 0.26 0.17 0 2523 Q27991 1353:1452 0.460.23 0.17 0 2524 — — 0.64 0.33 0.21 0 2525 P15989 398:447 0.57 0.41 0.180 2526 P02662  2:51 0.59 0.31 0.18 1 2527 P02662  3:52 0.59 0.33 0.18 12528 P02662  4:53 0.57 0.31 0.16 1 2529 P02662  5:54 0.55 0.29 0.14 12530 P02662  6:55 0.55 0.29 0.16 1 2531 P02662  79:128 0.41 0.28 0.12 02532 P02662  86:135 0.41 0.27 0.13 0 2533 Q9FRT9  1:90 0.32 0.11 0.07 102534 P09643  1:322 0.44 0.20 0.08 10 2535 Q9HD67 472:521 0.49 0.29 0.201 2536 Q02440 461:511 0.50 0.23 0.13 1 2537 Q13402 452:509 0.52 0.240.11 1 2538 Q02440 413:512 0.52 0.22 0.09 2 2539 Q28970 427:527 0.510.21 0.10 1 2540 Q90584 436:486 0.60 0.41 0.32 0 2541 P22281  82:1340.58 0.46 0.24 1 2542 Q17R14 270:321 0.64 0.39 0.20 0 2543 Q279911396:1446 0.49 0.31 0.23 0 2544 P12106 170:220 0.70 0.32 0.14 1 2545P32191 112:163 0.58 0.31 0.15 1 2546 P19524 1398:1449 0.56 0.25 0.18 22547 Q03262 225:277 0.65 0.30 0.13 1 2548 P12863  82:132 0.51 0.33 0.171 2549 P12106 159:209 0.67 0.29 0.14 1 2550 A6QR56 589:691 0.47 0.340.21 2 2551 P32492 1218:1319 0.67 0.25 0.14 1 2552 P79114 688:788 0.480.26 0.15 2 2553 Q5MIB5 492:592 0.53 0.27 0.14 2 2554 P04119  28:1290.51 0.26 0.17 2 2555 P06642  14:115 0.55 0.27 0.14 2 2556 Q2XQV4108:213 0.49 0.26 0.15 2 2557 P32492 1086:1287 0.58 0.29 0.15 1 2558Q76FS2  59:260 0.46 0.23 0.12 7 2559 Q36967  1:105 0.65 0.46 0.26 0 2560Q31721  1:58 0.63 0.38 0.19 2 2561 Q5ZMN0 44:93 0.53 0.36 0.26 0 2562Q5ZMN0 46:95 0.54 0.36 0.26 0 2563 Q5ZMN0 47:96 0.52 0.36 0.26 0 2564Q5ZMN0  1:146 0.53 0.30 0.21 2 2565 Q5ZMN0  1:147 0.52 0.30 0.21 2 2566Q5ZMN0  1:148 0.53 0.30 0.21 2 2567 Q5ZMN0  1:149 0.52 0.29 0.21 2 2568Q5ZMN0  1:150 0.52 0.29 0.21 2 2569 Q5ZMN0  1:151 0.52 0.29 0.20 2 2570Q5ZMN0  1:152 0.51 0.29 0.20 2 2571 Q5ZMN0  1:153 0.51 0.29 0.20 2 2572Q5ZMN0  1:154 0.51 0.28 0.20 2 2573 Q5ZMN0  1:161 0.49 0.27 0.19 2 2574Q9JLT0 127:231 0.48 0.21 0.15 0 2575 Q27991 141:190 0.51 0.28 0.23 02576 Q27991 136:185 0.51 0.28 0.21 0 2577 Q27991 116:185 0.51 0.27 0.210 2578 Q27991 146:200 0.51 0.29 0.21 0 2579 Q27991 146:210 0.52 0.270.20 0 2580 Q27991 136:190 0.50 0.27 0.21 0 2581 Q27991 146:195 0.500.30 0.21 0 2582 Q27991 126:190 0.52 0.26 0.19 0 2583 Q27991 141:2000.51 0.28 0.21 0 2584 Q27991 161:210 0.50 0.24 0.19 0 2585 Q27991126:185 0.53 0.27 0.19 0 2586 Q27991 111:210 0.50 0.26 0.19 0 2587Q27991 116:215 0.50 0.26 0.18 0 2588 Q27991 126:225 0.46 0.22 0.15 02589 Q27991 126:237 0.45 0.21 0.15 0 2590 Q9JLT0 194:244 0.45 0.20 0.140 2591 Q61879 154:252 0.41 0.19 0.14 0 2592 P15989  99:208 0.52 0.370.17 0 2593 P15989  99:203 0.51 0.37 0.16 0 2594 P15989  49:166 0.440.28 0.15 0 2595 Q90339 201:265 0.45 0.21 0.18 0 2596 Q9BE41 216:2650.48 0.21 0.18 0 2597 Q9TV62 241:290 0.48 0.17 0.15 0 2598 Q5SX39236:285 0.45 0.19 0.17 0 2599 Q9TV61 151:265 0.52 0.21 0.16 0 2600Q27991  20:289 0.38 0.20 0.15 0 2601 Q0WVK7  53:105 0.57 0.27 0.12 22602 Q94A52 149:261 0.54 0.26 0.12 2 2603 P38111 1093:1165 0.59 0.300.13 3 2604 P38111 1093:1182 0.57 0.27 0.13 3 2605 P38111 1093:1162 0.570.27 0.13 3 2606 P38111 1092:1166 0.59 0.29 0.13 3 2607 P38111 1093:11680.58 0.29 0.13 3 2608 P38111 1091:1164 0.59 0.30 0.13 3 2609 P381111089:1164 0.58 0.29 0.13 3

TABLE 8B Aller- Tox- Anti- Human Seq ID No SolvScore AggScore genicityicity nutricity Homology 2519 −15.79 0.80 0.31 0.00 1.00 0.67 2520−16.50 0.84 0.29 0.00 1.00 0.62 2521 −21.97 1.00 0.30 0.00 0.25 0.392522 −28.44 0.16 0.36 0.00 0.00 0.84 2523 −36.33 0.16 0.33 0.00 0.000.87 2524 −27.36 0.49 0.34 0.00 0.30 0.41 2525 −17.59 0.99 0.20 0.001.00 0.64 2526 −17.98 0.90 0.63 0.00 0.00 0.44 2527 −16.59 0.95 0.630.00 0.32 0.44 2528 −16.79 0.87 0.63 0.00 0.33 0.44 2529 −18.37 0.770.63 0.00 0.30 0.42 2530 −18.37 0.71 0.63 0.00 0.32 0.40 2531 −24.840.25 0.63 0.26 0.00 0.35 2532 −26.10 0.22 0.63 0.26 0.00 0.33 2533−15.45 0.39 0.32 1.00 0.34 0.34 2534 −19.21 0.44 0.99 0.00 0.00 0.962535 −25.30 0.46 0.22 0.33 0.34 1.00 2536 −22.49 0.43 0.21 0.31 0.350.94 2537 −22.06 0.51 0.21 0.28 0.27 1.00 2538 −21.01 0.41 0.31 0.000.28 0.94 2539 −23.16 0.43 0.28 0.00 0.00 0.98 2540 −15.02 1.17 0.220.34 0.35 0.75 2541 −10.60 1.38 0.22 0.00 0.34 0.28 2542 −18.91 0.890.20 0.00 0.32 0.94 2543 −31.57 0.29 0.21 0.00 0.31 0.88 2544 −17.130.77 0.20 0.31 0.28 0.61 2545 −16.98 0.76 0.19 0.25 0.17 0.52 2546−20.09 0.24 0.21 0.29 0.30 0.43 2547 −19.93 0.52 0.22 0.32 0.32 0.332548 −16.57 0.79 0.58 0.33 0.24 0.59 2549 −16.05 0.65 0.20 0.28 0.310.65 2550 −17.15 0.83 0.30 0.30 0.00 0.84 2551 −21.64 0.55 0.26 0.260.26 0.00 2552 −24.45 0.54 0.32 0.24 0.25 0.91 2553 −23.71 0.37 0.300.27 0.29 0.93 2554 −22.23 0.52 0.74 0.00 0.19 0.44 2555 −18.41 0.580.29 0.29 0.25 0.78 2556 −17.59 0.58 0.60 0.00 0.23 0.98 2557 −19.980.57 0.31 0.00 0.24 0.00 2558 −17.02 0.48 0.53 0.00 0.22 0.88 2559 −5.921.63 0.30 0.00 0.27 0.61 2560 −6.10 1.53 0.21 0.00 0.26 0.40 2561 −18.690.54 0.18 0.24 0.35 0.84 2562 −17.31 0.54 0.20 0.24 0.33 0.84 2563−17.40 0.47 0.20 0.22 0.34 0.84 2564 −22.65 0.39 0.32 0.25 0.25 0.762565 −22.50 0.39 0.32 0.25 0.25 0.76 2566 −22.35 0.39 0.32 0.25 0.260.76 2567 −22.74 0.39 0.32 0.00 0.25 0.76 2568 −22.59 0.39 0.32 0.000.25 0.75 2569 −22.97 0.38 0.32 0.24 0.25 0.76 2570 −23.34 0.38 0.320.24 0.25 0.75 2571 −23.25 0.38 0.32 0.24 0.25 0.75 2572 −23.61 0.370.32 0.00 0.25 0.75 2573 −24.11 0.36 0.32 0.23 0.26 0.75 2574 −33.590.15 0.34 0.25 0.00 1.00 2575 −36.26 0.21 0.21 0.29 0.33 0.92 2576−34.27 0.27 0.22 0.25 0.00 0.90 2577 −32.73 0.22 0.25 0.31 0.00 0.872578 −34.50 0.23 0.21 0.00 0.00 0.89 2579 −34.08 0.25 0.26 0.00 0.000.91 2580 −34.67 0.22 0.23 0.25 0.00 0.91 2581 −33.39 0.29 0.20 0.000.31 0.88 2582 −37.57 0.19 0.25 0.31 0.00 0.92 2583 −35.42 0.21 0.240.21 0.00 0.90 2584 −33.68 0.26 0.23 0.00 0.00 0.92 2585 −37.47 0.230.25 0.00 0.00 0.92 2586 −33.38 0.20 0.31 0.00 0.00 0.87 2587 −32.780.18 0.33 0.27 0.29 0.88 2588 −36.89 0.16 0.35 0.00 0.00 0.92 2589−37.66 0.15 0.39 0.00 0.00 0.93 2590 −29.21 0.16 0.25 0.00 0.27 1.002591 −30.24 0.12 0.33 0.00 0.00 0.99 2592 −16.02 0.94 0.32 0.00 1.000.63 2593 −16.69 0.94 0.30 0.00 1.00 0.63 2594 −17.69 0.65 0.31 0.001.00 0.62 2595 −34.37 0.07 0.29 0.00 0.00 0.98 2596 −38.56 0.08 0.240.00 0.00 0.98 2597 −41.59 0.04 0.26 0.00 0.00 0.98 2598 −40.29 0.040.23 0.28 0.00 0.98 2599 −35.70 0.11 0.36 0.26 0.22 0.99 2600 −34.970.13 0.40 0.23 0.22 0.93 2601 −26.91 0.45 0.18 0.30 0.22 0.31 2602−25.52 0.46 0.29 0.00 0.25 0.81 2603 −23.00 0.80 0.27 0.00 0.24 0.262604 −20.82 0.80 0.27 0.00 0.25 0.27 2605 −23.98 0.70 0.25 0.27 0.250.27 2606 −22.45 0.79 0.27 0.27 0.25 0.25 2607 −22.10 0.80 0.27 0.280.24 0.25 2608 −22.75 0.77 0.28 0.31 0.25 0.25 2609 −23.28 0.71 0.280.28 0.25 0.24

Example 7: Scaled Up Production of Recombinant Nutritive Proteins

A representative protocol for producing quantities of nutritive proteinsas described in this disclosure is as follows

5 ml LB with 100 mg/l carbenicillin (in a 50 ml baffled Pyrex shakeflask) is inoculated with a stab from the glycerol stock of arecombinant E. coli strain comprising a recombinant gene encoding anutritive protein and grown until late exponential phase (OD600 nm≈2) at37° C. and 250 rpm. A 2.51 Ultra Yield Flask (Thomson InstrumentCompany) is inoculated with 500 ml sterile water and enough EnBaseEnPresso™ tablets (BioSilta) to formulate 500 ml growth medium. Thismedium is supplemented with 100 mg/l carbenicillin, 0.001% Industrol 204antifoam, and 0.6 U/l EnzI'm (BioSilta). The shake flask is inoculatedto OD600 nm=0.05 and grown 16 hr at 30° C. and 250 rpm. At this point,OD600 nm z 10 had been reached, the growth medium is furthersupplemented with EnPresso™ Booster tablets (BioSilta), 1.2 U/1 EnzI'm,and 1 mM IPTG to induce heterologous protein production. After another8-24 hr of shaking at 30° C. and 250 rpm, the flask is harvested bycentrifugation at 10,000 rpm, the supernatant is decanted, and the wetcell weight is measured. At this point, approximately 20 gWCW (grams wetcell weight)/l medium is recovered.

The harvested cells from each shake-flask fermentation are suspended in25 mL of IMAC Equilibration Solution (30 mM Imidazole, 50 mM Phosphate,0.5 M NaCl, pH 7.5). The suspended cells are then lysed by sonication at90 Watts for 90 seconds, on ice. The lysed cells are centrifuged at15,500 RCF for 60 minutes, and decanted. The cell debris is discarded,and the supernatants are 0.2 μm filtered. Filters are then flushed withan additional 10 mL of IMAC Equilibration Solution. These filteredprotein solutions are then purified by immobilized metal affinitychromatography (IMAC).

IMAC resin (GE Healthcare, IMAC Sepharose 6 Fast Flow) is charged withnickel and equilibrated. 30 mL of each protein solution is loaded onto a5 mL IMAC column, and washed with additional equilibration solution toremove unbound impurities. The protein of interest is then eluted with15 mL of 0.5 M NaCl, 0.2 M Imidazole, pH 7.5. The purified proteins areshown to be 90% pure, by SDS-PAGE. Approximately 20 to 60 mg of eachprotein is recovered in the IMAC elution fractions. Each IMAC elutionfraction is buffer exchanged by dialysis into a formulation solution (20mM HEPES, pH 7.5). After buffer exchange, these protein solutions arerecovered for all downstream processing.

Example 8: Prediction of Soluble Expression of Nutritive Proteins

Open reading frames encoding a set of 292 nutritive proteins were clonedand introduced into E. coli to assess recombinant protein expressionusing the method of Example 6. In the system used, 163 proteins wereidentified as expressed while 129 were not. Of the 163 proteins thatexpressed, 125 were tested for soluble expression. It was found that 75were solubly expressed while 50 were not.

FIG. 1 shows a two dimensional histogram of protein expression in the E.coli expression screen. FIG. 1 shows the relative likelihood (on a logscale) of a protein being expressed as a function of solvation score(y-axis) and aggregation score (x-axis). A darker mark on the histogramindicates a higher number of proteins expressed, while a lighter markindicates a fewer number of proteins expressed. FIG. 1 shows that thoseproteins that were successfully expressed tend to cluster in the topleft region of the plot, where the solvation score is more negative(≦−20) and the aggregation score is smaller (≦0.75). There were fewexamples of proteins that were successfully expressed with less negativesolvation scores (≧−15) and large aggregation scores (≧1). This resultsuggests that nutritive proteins with solvation scores of −20 or lessand aggregation scores of 0.75 or less are more likely to be expressedin this system.

FIG. 2 shows a two dimensional histogram of the number of solubleprotein expression in the E. coli expression screen. FIG. 2 shows therelative likelihood (on a log scale) of a protein being solublyexpressed as a function of solvation score (y-axis) and aggregationscore (x-axis). Again, a darker mark on the histogram indicates a highernumber of proteins expressed, while a lighter mark indicates a fewernumber of proteins expressed. FIG. 1 shows that those proteins that wereexpressed solubly tended to cluster in the top left region of the plot,where the solvation score is more negative (≦−20) and the aggregationscore is smaller (≦0.5). There were few examples of proteins that wereexpressed solubly with less negative solvation scores (≧−15) and largeaggregation scores (≧0.75). This result suggests that nutritive proteinswith solvation scores of −20 or less and aggregation scores of 0.5 orless are more likely to be solubly expressed in this system.

Example 9: Solubility Screening

The solubility of nine nutritive proteins produced as described Examples6 and 7 was examined by centrifuge concentration followed by proteinconcentration assays. Samples in 20 mM HEPES pH 7.5 were tested forprotein concentration according to the protocol for Coomassie Plus(Bradford) Protein Assay (Thermo Scientific) and absorbance at 280 nm(if applicable). Based on these measurements 10 mg of protein was addedto an Amicon Ultra 3 kDa centrifugal filter (Millipore). Samples wereconcentrated by centrifugation at 10,000×g for 30 minutes. The finalconcentrated samples were examined for precipitated protein and color,and then tested for protein concentration as described above. Theresults are shown in Table 9.

TABLE 9 Seq ID No Appearance Concentration (g/L) 2580 Clear Faint Yellow44 2582 Clear Faint Yellow 166 2587 Clear Colorless 107 2595 ClearColorless 60 2596 Clear Colorless 29 2598 Clear Colorless 151 2599 ClearColorless 207 2600 Clear Yellow 95 2603 Clear Yellow 191

The solubilities of these nutritive proteins were found to besignificantly higher than concentrations typically found for whey (12.5g/L) and soy (10 g/L) (Pelegrine, D. H. G. & Gasparetto, C. A., 2005.Whey proteins solubility as function of temperature and pH. LWT—FoodScience and Technology, p. 77-80; Lee, K. H., Ryu, H. S. & and Rhee, K.C., 2003. Protein solubility characteristics of commercial soy proteinproducts. Journal of the American Oil Chemists' Society, pp. 85-90).This demonstrates the usefulness of the nutritive proteins disclosedherein. For example, the solubility of nutritive proteins may improvecompliant delivery of high quality protein in as small of a volume aspossible while avoiding the “chalkyness” that often characterizesproteins delivered in this manner. This may, for example, be useful todeliver proteins to the elderly or other subjects.

Example 10: Stability Screening

Thermal stability of nutritive proteins provides insight regardingwhether the protein is likely to have a useful shelf life. Samples ofproteins produced as described in Examples 6 and 7 were screened inparallel using a rapid thermal stability screening method. In thismethod proteins were heated slowly from 25° C. to 95° C. in tworepresentative formulations in the presence of a hydrophobic dye (EnzoLife Sciences, ProteoStat® Thermal shift stability assay kit) that bindsto aggregated proteins that form as the protein denatures withincreasing temperature (Niesen, F. H., Berglund, H. & Vadadi, M., 2007.The use of differential scanning fluorimetry to detect ligandinteractions that promote protein stability. Nature Protocols, Volume 2,pp. 2212-2221.). Upon binding, the dye's fluorescence increasessignificantly, which is then recorded by the rtPCR instrument andrepresented as the protein's melting curve (Lavinder, J. J., Hari, S.B., Suillivan, B. J. & Magilery, T. J., 2009. High-Throughput ThermalScanning: A General, Rapid Dye-Binding Thermal Shift Screen for ProteinEngineering. Journal of the American Chemical Society, pp. 3794-3795.).After the thermal shift is complete samples were examined for insolubleprecipitates and further analyzed by analytical size exclusionchromatography (SEC).

Protein solutions (12.5 mg/ml) were prepared in both PBS and 20 mM HEPESpH 7.7 buffers, each containing 1× ProteoStat TS Detection Reagent.Samples of each solution were heated slowly from 25° C.-95° C., 0.5°C./30 seconds using a real-time PCR (rtPCR) thermocycler whilemonitoring the fluorescence of the dye. From this thermal scan thetemperature of aggregation was determined (T_(agg)) from the temperaturewith the strongest slope if an increase in fluorescence was observed. Tosupplement the assay, samples were taken before and after the thermalshift and analyzed by SEC (GE Healthcare—Superdex 75 5/150) which candetect large soluble aggregates. The results for seven nutritiveproteins of this disclosure and a whey standard are presented in Table10. The presence of soluble aggregates detected by SEC is noted by a“yes” if observed or “no” if not observed, and the “n/a” entries for thewhey standard indicate the production of insoluble precipitates suchthat no SEC analysis was performed.

TABLE 10 Seq ID HEPES- PBS- HEPES-SEC PBS-SEC No T_(agg) T_(agg) Agg?Agg? 2582 95 95 No No 2587 95 95 No No 2596 45 95 No No 2598 95 95 No No2599 95 95 No No 2600 95 95 Yes Yes 2603 95 95 Yes Yes whey 79 81.5 n/an/a

As shown in Table 10, SEQ ID NOS: 2582, 2587, 2596, 2598, and 2599 hadmuch higher HEPES T_(agg) than whey (in fact, the proteins did not formany aggregates at 95° C., which was the upper limit of the assay), andthus are expected to be more stable than whey. The SEQ ID NOS: 2600 and2603 proteins had higher HEPES T_(agg) and PBS T_(agg) values than whey,but both formed soluble aggregates in solution while whey formedinsoluble aggregates that precipitated from solution. Thus, even in anaggregated state, these two nutritive proteins would be expected toremain in solution for a greater period of time than whey.

Example 11: Digestibility Screening—Determination of Digestion Half-Life

The goal of screening for protein digestibility is to eliminatepotentially unsafe allergenic proteins and to determine the relativecompleteness of digestion as a predictor of peptide bioavailability.This screening method utilizes a physiologically relevant in vitrodigestion reaction that includes both phases of protein digestion,simulated gastric digestion and simulated intestinal digestion (Moreno,J. F. et al., 2005. Stability of the major allergen Brazil nut 2Salbumin (Ber e 1) to physiologically relevant in vitro gastrointestinaldigestion. FEBS Journal, pp. 341-352.). Samples can be taken throughoutthe reaction and analyzed for intact protein and peptide fragments usingchip electrophoresis and LC-QTOF-MS. Proteins with allergenic propertiescan be assessed by identifying proteins or large fragments of proteinsthat are resistant to digestive proteases and thus have a higher risk ofcausing an allergenic reaction (Goodman, R. E. et al., 2008.Allergenicity assessment of genetically modified crops—what makessense?. Nature Biotechnology, pp. 73-81.). Digestibility is measured bydetermining how efficiently the protein is broken down into peptides(Daniel, H., 2003. Molecular and Integrative Physiology of IntestinalPeptide Transport. Annual Review of Physiology, Volume 66, pp.361-384.).

The method used an automated assay for in vitro digestions of proteinswherein assay conditions and protease concentrations are physiologicallyrelevant (Moreno, F. J., Mackie, A. R. & Clare Mills, E. N., 2005.Phospholipid interactions protect the milk allergen a-Lactalbumin fromproteolysis during in vitro digestion. Journal of agricultural and foodchemistry, pp. 9810-9816; Martos, G., Contreras, P., Molina, E. &Lopez-Fandino, R., 2010. Egg White Ovalbumin Digestion MimickingPhysiological Conditions. Journal of Agricultural and food chemistry,pp. 5640-5648; Moreno, J. F. et al., 2005. Stability of the majorallergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant invitro gastrointestinal digestion. FEBS Journal, pp. 341-352.). The firstphase of digestion is in simulated gastric fluid (SGF) and formulated atpH 1.5 and with a pepsin:substrate ratio of (1:10 w/w). The second phaseof digestion is in simulated intestinal fluid (SIF) is formulated withbile salts at pH 6.5 and with an trypsin:chymotrypsin:substrate ratio of(1:4:400 w/w). The protein is treated for 120 mins in the simulatedgastric fluid, which is how long it takes for 90% of a liquid meal topass from the stomach to the small intestine (Kong, F. & Singh, R. P.,2008. Disintegration of Solid Foods in Human Stomach. Journal of FoodScience, pp. 67-80), and then treated with simulated intestinal fluidfor 120 mins. Sample time points are taken throughout both reactions andquenched for analysis. Bovine serum albumin, which is readily digestedby pepsin, is the positive control for the SGF solution, andbeta-lactoglobulin, which is naturally resistant to pepsin but digestedin SIF, is the positive control for SIF solution. Intact protein andlarge fragments were detected using electrophoresis. For chipelectrophoresis, a Caliper Labchip GXII equipped with a HT Low MWProtein Assay Kit was used to monitor the size and amount of intactprotein as well as any digestion fragments larger than 4 kDa. Bymonitoring the amount of intact protein observed over time, thehalf-life (τ_(1/2)) of digestion was calculated for SGF and, if intactprotein is detected after SGF digestion, in SIF.

This method was used to analyze the digestion half-lives of thirteennutritive proteins of this disclosure (SEQ ID NOS: 2547, 2575, 2578,2580, 2582, 2587, 2595, 2596, 2598, 2599, 2600, 2602, and 2603) producedas described in Examples 6 and 7, as well as native and recombinantovalbumin (OVA and rOVA, respectively; SEQ ID NO: 2610) andbeta-lactoglobulin (BLG and rBLG, respectively; SEQ ID NO: 2611)proteins and a whey standard. The results of these experiments aresummarized in Table 11. An “n/a” entry in the Simulated Intestinal Fluidfield indicates that no intact protein was detected after SGF digestion.

TABLE 11 Digestion τ_(1/2) (min.) Simulated Simulated Seq ID No. GastricFluid Intestinal Fluid 2547 0.3 n/a 2575 0.3 n/a 2578 2 n/a 2580 6 n/a2582 0.5 n/a 2587 0.7 n/a 2595 10 n/a 2596 0.6 n/a 2598 0.7 n/a 2599 0.3n/a 2600 29 1 2602 1 n/a 2603 6 n/a BLG (2611) 77 4 rBLG (2611) 50 0.7OVA (2610) 18 1 rOVA (2610) 5 n/a whey 99 4

The results shown in Table 11 indicate that twelve of the thirteennutritive proteins of this disclosure were all completely digested bySGF and have SGF half lives of ten minutes or less. By comparison wheyis not completely digested by SGF and has an SGF half life of 99 minutesand a SIF half life of 4 minutes. This study suggests that the nutritiveproteins of this disclosure are likely to be readily digested and notlikely to elicit an allergic response when ingested.

The results in Table 11 also show that the recombinantbeta-lactoglobulin and ovalbumin produced according to this disclosurewere both more readily digested than their naturally-occurringcounterparts. The speed in which a protein is broken down can becontrolled by selecting for properties that improve or limitaccessibility of the gastrointestinal proteases. This capability can bedemonstrated for two typical protein properties, glycosylation anddisulfide cross-linking. Like many naturally occurring proteins,naturally occurring OVA and BLG are glycosylated by their hostorganisms. In contrast, the recombinant proteins produced according tothe present disclosure are not glycosylated because the host organism(E. coli in this case) does not glycosylate. The lack of glycosylationin recombinant nutritive proteins according to this disclosure mayresult in proteins that are more readily digested. Furthermore, BLG hasfour disulfide bonds that are known to slow down or interfere withdigestion. When these disulfide bonds are disrupted, the rate ofdigestion increases (Reddy, I. M., Kella, N. K. D. & Kinsella, J. E.,1988. Structural and conformational Basis of the Resistance ofb-Lactoglobulin to Peptic and Chymotryptic Digestion. J. Agric. FoodChem., Volume 36, pp. 737-741). A lack or disruption of disulfide bondformation in recombinant nutritive proteins according to this disclosuremay result in proteins that are more readily digested.

Example 12: Digestibility Screening—Analysis of Digestion Products

Two nutritive proteins produced as described in Examples 6 and 7 (SEQ IDNOS: 2612 and 2613) were subjected to SGF and SIF digestion as describedin Example 11. Both proteins were completely digested in SGF, and theSGF half lives are shown in Table 12.

TABLE 12 Digestion τ_(1/2) (min.) Seq ID No Simulated Gastric FluidSimulated Intestinal Fluid 2612 0.7 n/a 2613 6 n/a

To detect and identify peptides that were present after SGF and SIFdigestion, samples of the SGF and SIF digests were analyzed by LC/Q-TOFMS/MS. Samples from the SGF digests were directly analyzed by LC/Q-TOFMS/MS, while SIF protein digestions required purification by SCX toremove bile acids before detection and identification by LC/Q-TOF MS/MS.Peptides were extracted from the chromatograms and identified usingBioconfirm Software (Aglient). The sequence assignment of peptides werebased on accurate mass match (±10 ppm) and further confirmed by MS/MSfragmentation. The results are shown in Tables 13 and 14 below.

TABLE 13 (SEQ ID NO: 2612) SEQ SEQ SGF Peptides ID SIF Peptides ID120 min NO 120 min NO LL SE LAL PSE HVL HVL LEL FKV LALA 2616 HQI LLLD2617 PSEA 2618 IAEF 2619 REV IQQF 2620 FDK YDKL 2621 AEFK 2622 SNLTE2623 LKHV 2624 ELLEA 2625 SSSEL 2626 EELAL 2627 FKVF 2628 DDLLL 2629AELKH 2630 LAYDK 2631 LKHVL 2632 TKTRL 2633 FKEAF 2634 DLDHQ 2635NGSISSS 2636 GTLENL 2637 SLGLSPS 2638 EKLTDA 2639 GEKLTD 2640 AEVDDM2641 ELATVM 2642 LDDLLL 2643 GSGEINI 2644 LDLDHQ 2645 RSLGLSP 2646DLKKKL 2647 ELKHVL 2648 KTKTRL 2649 KLTDAEV 2650 SQRLEE 2651 FKVFDK 2652AEVDDML 2653 AEVDDML 2654 QQELDDL 2655 DAEVDDM 2656 LEKTKTR 2657 AELKHVL2658 LEKTKTR 2659 HVLTSIGE 2660 GTLENLEE 2661 SIGEKLTD 2662 QQELDDLL2663 RSLGLSPSE 2664 KLEKTKTRLQ 2665 VLTSIGEKL 2666 SRQLKSNDSEQ 2667TSIGEKLTD 2668 EKTKTRLQQEL 2669 HVLTSIGEK 2670 YDKLEKTKTRL 2671AELKHVLTS 2672 LAYDKLEKTKTRL 2673 RSLGLSPSEA 2674 EELKKKLLKDLEL 2675SSNLTEEQIA 2676 EELKKKLLKDLELL 2677 RSLGLSPSEAE 2678 AELKHVLTSIGEKLTD2679 KVFDKNGDGLISA 2680 MGSHHHHHHHHSSNL 2681 FDKDNNGSISSSEL 2682LREVSDGSGEINIQQF 2683 REVSDGSGEINIQQ 2684 AAELKHVLTSIGEKLTD 2685DVDGNHQIEFSEF 2686 AELKHVLTSIGEKLTDAE 2687 LREVSDGSGEINIQQ 2688AYDKLEKTKTRLQQEL 2689 REVSDGSGEINIQQF 2690 AAELKHVLTSIGEKLTDAE 2691LREVSDGSGEINIQQ 2692 FAALLS LENLEELKKKLLKDLEL 2693 KLEKTKTRLQQELDDLL2694 DKLEKTKTRLQQELDDLL 2695 LAYDKLEKTKTRLQQELDDL 2696LALAYDKLEKTKTRLQQELDDL 2697

TABLE 14 (SEQ ID NO: 2613) SGF Peptides SEQ ID SIF Peptides SEQ ID120 min NO 120 min NO GVL TKH ALL INDI 2698 LVL HLVL 2699 IGVL 2700 TIKF2701 TIKF 2702 IGVLD 2703 TIKF 2704 RNLD 2705 EVYDL 2706 IGVLDV 2707LNDSVQ 2708 QTIKF 2709 IWVIND 2710 VQTIKF 2711 DLNDSVQ 2712 SVQTIKF 2713SVQTIKF 2714 KCAKCISMIGVL 2715 HHLVLGALLD 2716 EKCAKCISMIGV 2717HHHHHHLVL 2718 HEFKRTTYSE 2719 HHHHHHHHLVL 2720 SHKFRNLDKDL 2721DVTKHEFKRTTY 2722 ISMIGVLDVTKHE 2723 SHHHHHHHHLVL 2724 TKHEFKRTTYSEN2725 GSHHHHHHHHLVLG 2726 MGSHHHHHHHHLVL 2727 KRTTYSENEVYDLN 2728

As can been seen in Tables 13 and 14, each protein was digested intomultiples smaller peptide fragments ranging in size from 2 to 22 aminoacids (SEQ ID NO: 2612) or 2 to 13 amino acids (SEQ ID NO: 2613). Noneof these peptide fragments was found to be homologous to any knownallergen.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An isolated nutritive protein comprising a first polypeptidecomprising a first polypeptide sequence that is homologous to a fragmentof a naturally-occurring protein, wherein the fragment is at least 25amino acids in length, wherein the first polypeptide sequence comprisesat least one of: a. a ratio of branch chain amino acid residues to totalamino acid residues of at least 24%; b. a ratio of Leu residues to totalamino acid residues of at least 11%; and c. a ratio of essential aminoacid residues to total amino acid residues of at least 49%, and whereinthe first polypeptide sequence has less than 90% global homology to aknown allergen.
 2. The isolated nutritive protein of claim 1, whereinthe first polypeptide sequence further comprises at least one of eachessential amino acid. 3.-4. (canceled)
 5. The isolated nutritive proteinof claim 1, wherein the fragment of a naturally-occurring proteincomprises at least 25 amino acid residues.
 6. The isolated nutritiveprotein of claim 1, wherein the fragment of a naturally-occurringprotein comprises at least 50 amino acid residues.
 7. The isolatednutritive protein of claim 1, wherein the first polypeptide sequencecomprises: a. a ratio of branch chain amino acid residues to total aminoacid residues of at least 24%; b. a ratio of Leu residues to total aminoacid residues of at least 11%; and c. a ratio of essential amino acidresidues to total amino acid residues of at least 49%. 8.-69. (canceled)70. An isolated nucleic acid comprising a nucleic acid sequence thatencodes a nutritive protein according to claim
 1. 71.-74. (canceled) 75.A vector comprising a nucleic acid sequence that encodes a nutritiveprotein of claim
 1. 76. (canceled)
 77. A recombinant microorganismcomprising the nucleic acid of claim
 70. 78.-81. (canceled)
 82. A methodof making a nutritive protein, the method comprising culturing arecombinant microorganism according to claim 77 under conditionssufficient for production of the nutritive protein by the recombinantmicroorganism.
 83. (canceled)
 84. A nutritive composition comprising anutritive protein of claim 1 and at least one second component. 85.-100.(canceled)
 101. A method of making a nutritive composition, comprisingproviding a nutritive protein according to claim 1 and combining thenutritive protein with the at least one second component.
 102. A methodof maintaining or increasing at least one of muscle mass, musclestrength, and functional performance in a subject, the method comprisingproviding to the subject a sufficient amount of a nutritive protein ofclaim
 1. 103. A method of maintaining or achieving a desirable body massindex in a subject, the method comprising providing to the subject asufficient amount of a nutritive protein of claim
 1. 104. (canceled)105. A method of providing protein to a subject with protein-energymalnutrition, the method comprising providing to the subject asufficient amount of a nutritive protein of claim
 1. 106. A method ofincreasing thermogenesis in a subject, the method comprising providingto the subject a sufficient amount of a nutritive protein of claim 1.107. A method of inducing at least one of a satiation response and asatiety response in a subject, the method comprising providing to thesubject a sufficient amount of a nutritive protein of claim
 1. 108.-109.(canceled)
 110. A method of treating at least one of cachexia,sarcopenia and frailty in a subject, the method comprising providing tothe subject a sufficient amount of a nutritive protein of any one ofclaim
 1. 111. (canceled)
 112. A method of making a nutritive protein ofclaim 1, the method comprising chemically synthesizing the protein. 113.A method of making a nutritive protein of claim 1, the method comprisingisolating the protein.
 114. The isolated nutritive protein of claim 1,wherein the first polypeptide sequence is at least 25 amino acids inlength.